AU2019201141B2

AU2019201141B2 - Novel antibody binding to TFPI and composition comprising the same

Info

Publication number: AU2019201141B2
Application number: AU2019201141A
Authority: AU
Inventors: Ki Joon Cho; Sungho HWANG; Junhong JEONG; Dong-Sik Kim; Kisu Kim; Heechun KWAK; Mi Jung Lee; Sumin Lee; Hyung-Kwon Lim; Jae Chan Park; Young Seoub Park
Original assignee: Mogam Institute for Biomedical Research
Current assignee: Mogam Institute for Biomedical Research
Priority date: 2015-02-25
Filing date: 2019-02-19
Publication date: 2020-05-07
Anticipated expiration: 2035-12-29
Also published as: CN107428838A; EP3262075C0; CN112225812A; US10266607B2; CN112225812B; AU2015384281A1; JP2018511305A; BR112017018328A2; EA201791898A1; EP3262075A1; EP3262075A4; US20180030151A1; WO2016137108A1; CN107428838B; MY178445A; CA2977621A1; EP3262075B1; JP6660957B2; CA2977621C; EP4279128A3

Abstract

The present invention relates to an antibody that binds specifically to a tissue factor pathway inhibitor (TFPI), a nucleic acid encoding the antibody, a vector comprising the nucleic acid, a host cell transformed with the vector, a method for producing the antibody, 5 and a pharmaceutical composition for treating hemophilia, which comprises the antibody as an active ingredient. The antibody of the present invention, which binds specifically to TFPI, can activate the extrinsic pathway of blood coagulation by inhibiting TFPI. Thus, the antibody of the present invention can be effectively used for the treatment of antibody induced hemophilia patients and for the prevention of blood coagulation disease in 10 hemophilia-A or hemophilia-B patients.

Description

NOVEL ANTIBODY BINDING TO TFPI AND COMPOSITION COMPRISING THE SAME

Corresponding Applications This is a divisional of Australian Patent Application No. 2015384281, which is the Australian National Phase of PCT/KR2015/014370, which claims priority from Korean patent application numbers 10-2015-0135761, filed 25 September 2015 and 10-2015 0026555 filed 25 September 2015. All publications, patent applications, patents, and other references mentioned herein are incorporated by referenced in their entirety.

Technical Field The present invention relates to an antibody that binds specifically to a tissue

factor pathway inhibitor (TFPI), a nucleic acid encoding the antibody, a vector

comprising the nucleic acid, a host cell comprising the vector, a method for producing the

antibody, and a pharmaceutical composition for treating hemophilia, which comprises the

antibody as an active ingredient.

Background Art It is known that, in about 30% of patients with hemophilia A and B, an antibody

against the FVIII (factor VIII) or FIX (factor IX) protein used for treatment is produced

to significantly reduce the therapeutic effect of the protein. As an alternative to the

protein, activated factor VIIa or aPCC (plasma-derived activated prothrombin complex

concentrate) has been administered.

The above-described recombinant protein is administered to hemophilia patients

by intravenous injection twice or more a week, but inconvenience caused by repeated

administration of the recombinant protein has been constantly presented. Thus, studies on long-acting recombinant proteins having an increased half-life have been actively conducted.

In hemophilia models, an approach against TFPI (tissue factor pathway inhibitor)

has recently been attempted. TFPI is involved in the extrinsic pathway of blood

coagulation, and functions to inhibit blood coagulation by preventing factor X activation

with TF/FVIIa (see FIG. 1). Thus, when TFPI is inhibited by an anti-TFPI antibody,

blood coagulation during bleeding can be activated by the extrinsic pathway.

TFPI consists of three KPI domains (Kunitz-type domains or Kunitz domains),

and KPI-2 (Kunitz domain 2) inhibits FXa by binding directly to FXa (see FIG. 2). This

means that KPI-2 forms a complex of TF/FVIIa/FXa/TFPI, resulting in direct inhibition

of production of FXa.

An anti-TFPI antibody may be used in patients in which an antibody against the

FVIII or FIX protein has been produced. In addition, the anti-TFPI antibody has a very

long half-life (about 2 weeks), and thus the number of administrations thereof can be

reduced.

Hemophilia therapeutic agents against TFPI are mostly in the research stage or

the initial development stage. For example, the humanized monoclonal antibody (mAb)

mAb2021 developed by Novo Nordisk is a humanized antibody (IgG4) that is an anti

TFPI monoclonal antibody, and is in the phase 1 clinical stage. In addition, ARC19499

developed by Baxter is a PEGylated aptamer targeting TFPI and is in the preclinical stage.

Furthermore, JBT2329 developed by Baxter & 3B Pharmaceuticals is a Pegylated anti

human TFPI 20mer peptide and is in the preclinical stage.

The need for a new agent for treating hemophilia has been constantly proposed,

and the development of therapeutic agents that are approaches other than a bypassing

agent such as FVIIa is urgently required. In particular, an approach to a drug that inhibits the TFPI pathway is preferred. Among hemophilia patients who are administered with a blood coagulation factor, a number of patients having resistance to the factor exist, and thus require a new drug. However, medical issues such as antigen (Ag)-antibody (Ab) complex clearance should be taken into consideration.

Accordingly, the present inventors have made extensive efforts to develop a

novel antibody that binds specifically to TFPI, and as a result, have found that the use of

the antibody can activate the extrinsic pathway of blood coagulation by inhibiting the

anticoagulation mechanism of TFPI, thereby completing the present invention.

Disclosure Of Invention Technical Problem It is an object of the present invention to provide a novel antibody that binds

specifically to TFPI, a nucleic acid encoding the antibody, a vector comprising the

nucleic acid, a host cell comprising the vector, a method for producing the antibody, and

a pharmaceutical composition for treating antibody-induced hemophilia or preventing a

blood coagulation disorder in hemophilia-A and hemophilia-B patients, the

pharmaceutical composition comprising the antibody as an active ingredient and being

capable of inhibiting TFPI to thereby activate the extrinsic pathway of blood coagulation.

Technical Solution To achieve the above object, the present invention provides an antibody that

binds specifically to a TFPI (tissue factor pathway inhibitor) represented by SEQ ID NO:

165.

The present invention also provides: a nucleic acid encoding an anti-TFPI antibody;

a vector containing the nucleic acid; and a cell having the vector introduced therein.

The present invention also provides a pharmaceutical composition for treating

hemophilia, which comprises an anti-TFPI antibody as an active ingredient.

Throughout this specification the word "comprise", or variations such as

"comprises" or "comprising", will be understood to imply the inclusion of a stated element,

integer or step, or group of elements, integers or steps, but not the exclusion of any other

element, integer or step, or group of elements, integers or steps.

Any discussion of documents, acts, materials, devices, articles or the like which has

been included in the present specification is not to be taken as an admission that any or all

of these matters form part of the prior art base or were common general knowledge in the

field relevant to the present disclosure as it existed before the priority date of each of the

appended claims.

Brief Description Of The Drawings FIG. 1 shows the extrinsic pathway of blood coagulation and TFPI.

FIG. 2 shows the schematic protein structure of TFPI and the function of KPI

domains.

FIG. 3 shows the results of protein electrophoresis (SDS-PAGE) of T417 and T308

clone antibodies among purified anti-TFPI antibodies.

FIG. 4 shows the amino acid sequences of clone T417 and humanized antibody

clone 308 among anti-TFPI antibodies.

FIG. 5 shows the amino acid sequences of 308-2 and 302-4 clone antibodies that

are clone 308 antibody mutants among anti-TFPI antibodies.

FIG. 6 shows the results of protein electrophoresis (SDS-PAGE) of IgG of 308-2

and 302-4 clone antibodies that are clone 308 antibody mutants among anti-TFPI antibodies.

FIG. 7 shows the results of protein electrophoresis (SDS-PAGE) of the TFPI KPI-2

(Kunitz domain 2) protein according to the type of animal.

FIG. 8 graphically shows the affinities of anti-TFPI antibodies.

FIG. 9 graphically shows the results of evaluating the effects of chimeric antibodies

among anti-TFPI antibodies by an FXa activity assay.

4A

FIG. 10 graphically shows the results of evaluating the effects of humanized

antibodies among anti-TFPI antibodies by an FXa activity assay.

FIG. 11 graphically shows the results of evaluating the effects of back-mutated

antibodies among anti-TFPI antibodies by an FXa activity assay.

FIG. 12 graphically shows the results of evaluating the effects of chimeric

antibodies among anti-TFPI antibodies by a TF/FVIIa/FXa complex assay.

FIG. 13 graphically shows the results of evaluating the effects of humanized

antibodies among anti-TFPI antibodies by a TF/FVIIa/FXa complex assay.

FIG. 14 graphically shows the results of evaluating the effects of back-mutated

antibodies among anti-TFPI antibodies by a TF/FVIIa/FXa complex assay.

FIG. 15 graphically shows the results of evaluating the effects of chimeric and

humanized antibodies among anti-TFPI antibodies by a thrombin generation assay.

FIG. 16 graphically shows the results of evaluating the effects of back-mutated

antibodies among anti-TFPI antibodies by a thrombin generation assay.

FIG. 17 shows the predicted binding between clone 308 among anti-TFPI

antibodies and a human TFPI K2 domain. The molecule indicated by red indicates the

human TFPI K2 domain, and the molecule indicated by green indicates the clone 308

antibody.

FIG. 18 shows the predicted binding between the heavy-chain variable region of

clone 308 among anti-TFPI antibodies and a human TFPI antigen.

FIG. 19 shows the predicted binding between the light-chain variable region of

clone 308 among anti-TFPI antibodies and a human TFPI antigen.

FIGS. 20 to 28 show the results of evaluating the effects of affinity-matured anti

TFPI antibodies by an Fxa activity assay.

FIGS. 29 to 33 show the results of evaluating the effects of affinity-matured anti

TFPI antibodies by a TF/FVIIa/FX complex assay.

FIG. 34 shows the results of evaluating the effects of affinity-matured anti-TFPI

antibodies by a thrombin generation assay.

Best Mode For Carrying Out The Invention It was reported that TFPI (tissue factor pathway inhibitor) is involved in the

extrinsic pathway of blood coagulation and inhibits blood coagulation by preventing

factor X activation with TF/FVIIa. Thus, the present inventors have attempted to

construct an antibody for treating or preventing hemophilia. In particular, it is the subject

matter of the present invention to activate the extrinsic pathway of blood coagulation by

an antibody that inhibits the KPI-2 of TFPI. The antigen region that is targeted by the

antibody is the KPI-2 domain of TFPI, which has an amino acid sequence having an

identity of 90% or higher between humans, rabbits and monkeys. Thus, when the KPI-2

domain of TFPI is used, an animal study is easily designed, and a simple model for

measuring the rate of blood coagulation can be introduced.

As used herein, the term "tissue factor pathway inhibitor" or "TFPI" refers to any

variant, isoform and species homolog of human TFPI that is naturally expressed by cells.

In a preferred embodiment of the invention, the binding of an antibody of the invention to

TFPI reduces the blood coagulation time.

In an example of the present invention, "clone 308", "clone 308-2", and "clone

308-4" were prepared, which are isolated human monoclonal antibodies having a

structural characteristic that binds specifically to a TFPI (tissue factor pathway inhibitor)

represented by SEQ ID NO: 39. The amino acid sequences of the heavy-chain CDR and

light-chain CDR of each of the antibodies are as shown in Tables 5 and 7 below. As

shown in Tables 4 and 6 below, anti-TFPI antibodies may comprise the amino acid sequences of a heavy-chain variable region and a light-chain variable region and sequences homologous thereto.

In another example of the present invention, the quantitative affinity of the

purified antibody clone T417, clone T308, clone 308, clone 308-2 or clone 308-4 for

recombinant human TFPI was measured using a Biacore T-200 biosensor (GE Healthcare,

USA) (Example 6). As a result, as shown in Table 13 and FIG. 8, all the prepared clone

antibodies affinities which were somewhat different from one another. Particularly, it

was shown that the affinities of clone 308-2 and clone 308-4 were very higher than that

of clone 308.

Thus, in one aspect, the present invention is directed to an antibody that binds

specifically to a TFPI (tissue factor pathway inhibitor) represented by SEQ ID NO: 39.

In the present invention, the antibody may contain a heavy-chain variable region

comprising: a heavy-chain CDR1 comprising an amino acid sequence of SEQ ID NO: 5,

11 or 23; a heavy-chain CDR2 comprising an amino acid sequence of SEQ ID NO: 6, 12,

26 or 27; and a heavy-chain CDR3 comprising an amino acid sequence of SEQ ID NO: 7

or 13.

comprising: a light-chain CDR1 comprising an amino acid sequence of SEQ ID NO: 8 or

14; a light-chain CDR2 comprising an amino acid sequence of SEQ ID NO: 9 or 15; and

a light-chain CDR3 comprising an amino acid sequence of SEQ ID NO: 10 or 16.

comprising a sequence having a homology of at least 80%, preferably at least 90%, more

preferably 100%, to an amino acid sequence of SEQ ID NO: 1, 3, 21, 24 or 25, and the

antibody may contain a light-chain variable region comprising a sequence having a homology of at least 80%, preferably at least 90%, more preferably 100%, to an amino acid sequence of SEQ ID NO: 2, 4 or 22.

comprising an amino acid sequence of SEQ ID NO: 1, 3, 21, 24 or 25, and a light-chain

variable region comprising an amino acid sequence of SEQ ID NO: 2, 4 or 22. The

antibody may be a human monoclonal antibody, but is not limited thereto.

In an example of the present invention, "clone 1001", "clone 1015", "clone 1021",

"clone 1023" "clone 1024", "clone 1104", "clone 1123", "clone 1202", "clone 1208",

"clone 1214", "clone 1216", "clone 1223", "clone 1224", "clone 1232", "clone 1234",

"clone 1238", "clone 1243", "clone 1248", "clone 3007", "clone 3016", "clone 3024",

"clone 3115", "clone 3120", "clone 3131", "clone 3203", "clone 3241", "clone 4011",

"clone 4017", "clone 4034", "clone 4041", "clone 4141", "clone 4146", "clone 4206",

"clone 4208", "clone 4278", "clone 4287", "clone 1", "clone 2", "clone 3", "clone 4",

"clone 5", "clone 6", "clone 7", "clone 8", "clone 9", "clone 10", "clone 11", "clone 12",

"clone 13", "clone 14", "clone 15", "clone 16", "clone 17", "clone 18", "clone 19", "clone

20", "clone 21", "clone 22", "clone 23", "clone A24", "clone A25", "clone A52", "clone

A63", "clone A67", "clone A71", and "clone A74" were prepared, which are isolated

monoclonal antibodies having a structural characteristic that binds specifically to a TFPI

(tissue factor pathway inhibitor) represented by SEQ ID NO: 39. The amino acid

sequences of the heavy-chain CDR and light-chain CDR of each of the antibodies are as

shown in Tables 20 and 23 below. As shown in Tables 19 and 22 below, anti-TFPI

antibodies may comprise the amino acid sequences of a heavy-chain variable region and

a light-chain variable region and sequences homologous thereto.

In another example of the present invention, the quantitative binding affinities of

clone 12, clone 1023, clone 1202 and clone 3241, which are purified antibodies, for recombinant human TFPI, were measured using a Biacore T-200 biosensor (GE

Healthcare, USA) (Example 13). As a result, as shown in Table 24 below, all the

prepared clone antibodies showed affinities which were somewhat different from one

another.

comprising: a heavy-chain CDR1 comprising an amino acid sequence of SEQ ID NO:

149, 157, 163, 172, 181, 182, 183, 188, 201 or 203; a heavy-chain CDR2 comprising an

amino acid sequence of SEQ ID NO: 150, 155, 159, 162, 165, 166, 167, 168, 173, 184,

186, 187 or 202; and a heavy-chain CDR3 comprising an amino acid sequence of SEQ ID

NO: 151, 156, 170, 174, 175 or 185.

comprising: a light-chain CDR1 comprising an amino acid sequence of SEQ ID NO: 152,

158, 160, 169, 171, 176, 177 or 178; a light-chain CDR2 comprising an amino acid

sequence of SEQ ID NO: 153; and a light-chain CDR3 comprising an amino acid

sequence of SEQ ID NO: 154, 161, 164, 179 or 180.

preferably 100%, to an amino acid sequence of SEQ ID NO: 95, 97, 98, 99, 100, 102, 104,

105,107,109, 110, 112, 113, 114,115, 117,118, 119,120, 121, 123,124, 125,126,127,

128,129,131,132,133,134,135,136,137,138,141,142,143,144,145,146,148,195,

197, 198, 199 or 200, and the antibody may contain a light-chain variable region

comprising a sequence having a homology of at least 80%, preferably at least 90%, more preferably 100%, to an amino acid sequence of SEQ ID NO: 96, 101, 103, 106, 108, 111,

116,122,130,139,140,147or196.

comprising an amino acid sequence of SEQ ID NO: 95, 97, 98, 99, 100, 102, 104, 105,

107,109,110,112,113,114,115,117,118,119,120,121,123,124,125,126,127,128,

129,131,132,133,134,135,136,137,138,141,142,143,144,145,146,148,195,197,

198, 199 or 200, and a light-chain variable region comprising an amino acid sequence of

SEQ ID NO: 96, 101, 103, 106, 108, 111, 116, 122, 130, 139, 140, 147 or 196. The

antibody may be a human monoclonal antibody, but is not limited thereto.

The amino acid sequence of the antibody can be replaced by conservative

substitution. As used herein, the term "conservative substitution" refers to modifications

of a polypeptide that involve the substitution of one or more amino acids for amino acids

having similar biochemical properties that do not result in loss of the biological or

biochemical function of the polypeptide. A "conservative amino acid substitution" is one

in which the amino acid residue is replaced with an amino acid residue having a similar

side chain. Families of amino acid residues having similar side chains have been defined

in the art to which the present invention pertains. These families include amino acids

(e.g., lysine, arginine and histidine with basic side chains, amino acids (e.g., aspartic acid

and glutamic acid) with acidic side chains, amino acids (e.g., glycine, aspargin, glutamine,

serine, threonine, tyrosine, and cysteine) with uncharged polar side chains, amino acids

(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and

tryptophan) with nonpolar side chains, amino acids (e.g., threonine, valine, and

isoleucine) with beta-branched side chains, and amino acids (e.g., tyrosine, phenylalanine,

tryptophan, and histidine) with aromatic side chains. It is envisioned that the antibodies of the present invention may have conservative amino acid substitutions and still retain activity.

For nucleic acids and polypeptides, the term "substantial homology" indicates

that two nucleic acids or two polypeptides, or designated sequences thereof, when

optimally aligned and compared, are identical, with appropriate nucleotide or amino acid

insertions or deletions, in at least about 80% of the nucleotides or amino acids, usually at

least about 85%, preferably at least about 90%, 91%, 92%, 93%, 94%, or 95%, more

preferably at least about 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5%

of the nucleotides or amino acids. Alternatively, substantial homology for nucleic acids

exists when the segments will hybridize under selective hybridization conditions to the

complement of the strand. Also included are nucleic acid sequences and polypeptide

sequences having substantial homology to the specific nucleic acid sequences and amino

acid sequences recited herein.

As shown in Tables 2, 5 and 7 below, in the antibodies according to the present

invention, the heavy-chain (VH)CDR1, CDR2 and CDR3 sequences and the light-chain

(VL) CDR1, CDR2 and CDR3 sequences may be composed of a mixture of structurally similar heavy-chain(VH) and light-chain (VL) sequences which form CDR1, CDR2 and

CDR3, each consisting of a heavy chain(VH)/light chain (VL) pair.

As shown in Tables 5 and 8 below, in the antibodies according to the present

CDR3, each consisting of a heavy chain(VH)/light chain (VL) pair.

As used herein, the term "antibody" or "antibody composition" refers to a

preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to antibodies displaying a single binding specificity that have variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present invention can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

As used herein, the term "antibody" refers to a protein molecule which comprises

an immunoglobulin molecule immunologically reactive with a particular antigen, and

which serves as a receptor that specifically recognizes an antigen. The term may include

all polyclonal antibodies, monoclonal antibodies, full-length antibodies, and antibody

fragments. In addition, the term may include chimeric antibodies (e.g., humanized

murine antibodies), bivalent or bispecific molecules (e.g., bispecific antibodies),

diabodies, triabodies and tetrabodies.

A full-length antibody has two full-length light chains and two full-length heavy

chains, in which each of the light chains is linked to the heavy chain by a disulfide bond.

The full-length antibody comprises IgA, IgD, IgE, IgM and IgG, and subtypes of IgG

include IgG1, IgG2, IgG3 and IgG4. The term "antibody fragment"refers to a fragment

having an antigen-binding function, and is intended to include Fab, Fab', F(ab')2, scFv

and Fv.

Fab comprises light-chain and heavy-chain variable regions, a light-chain

constant region, and a heavy-chain first constant domain (CH1), and has one antigen

binding site. Fab' differs from Fab in that it has a hinge region including one or more

cysteine residues at the C-terminus of the heavy-chain CHi domain. An F(ab')2 antibody

is formed by a disulfide bond between the cysteine residues of the hinge region of Fab'.

Fv (variable fragment) means a minimal antibody fragment having only a heavy

chain variable region and a light-chain variable region. dsFv is has a structure in which a

heavy-chain variable region and a light-chain variable region are linked to each other by a

disulfide bond, and scFV generally has a structure in which a heavy-chain variable region

and a light-chain variable region are covalently linked to each other by a peptide linker.

These antibody fragments can be obtained using proteases (for example, Fab fragments

can be obtained by digesting a full-length antibody with papain, and F(ab')2 fragments

can be obtained by digesting a full-length antibody with pepsin). Preferably, these

antibody fragments can be produced by a genetic recombinant technique (for example,

performing amplification by PCR (polymerase chain reaction) using as a template a DNA

encoding the heavy chain of the antibody or the variable region thereof and a DNA

encoding the light chain or the variable region thereof together with a primer pair, and

performing amplification using a combination of primer pairs such that a DNA encoding

a peptide linker is connected with the heavy chain or the variable region thereof and the

light chain and the variable region thereof).

An immunoglobulin has heavy chains and light chains, and each heavy and light

chain contains a constant region and a variable region (the regions are also known

domains). Light and heavy chain variable regions contain four framework regions and

three hypervariable regions, also called "complementarity-determining regions"

(hereinafter referred to as "CDRs"). The CDRs are primarily responsible for binding to

an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1,

CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also

typically identified by the chain in which the particular CDR is located.

The terms "monoclonal antibody", as used herein, refers to antibody molecules

having a single molecular composition, obtained from a population of essentially identical antibodies. This monoclonal antibody can display a single binding specificity and affinity for a particular epitope.

As used herein, the term "monoclonal antibody" refers to a molecule derived

from human immunoglobulin, in which the full-length amino acid sequence of the

antibody, including complementarity-determining regions and framework regions,

consists of the amino acid sequence of human immunoglobulin. Human antibodies are

generally used for the treatment of human diseases and have the following advantages.

First, the human antibody can more easily interact with the human immune system so that

target cells can be more efficiently destroyed by, for example, complement-dependent

cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). Second,

the human immune system does not recognize the antibody as an external antibody.

Third, even when the antibody is administered in a smaller mount at a lower frequency,

the half-life thereof in the human circulatory system is similar to that of a naturally

occurring antibody.

Thus, the antibody according to the present invention is a monoclonal antibody

that binds specifically to TFPI, and can show a high affinity and specificity for TFPI. In

addition, because the antibody of the present invention is of human origin, it shows low

immunogenicity, and thus is effectively used for the treatment of diseases such as

antibody-induced hemophilia (hemophilia-A or hemophilia-B).

As used herein, the term "clone T417", "clone T308", "clone 308", "clone 308-2"

or "clone 308-4" that binds specifically to TFPI means an antibody that binds to TFPI,

resulting in inhibition of the biological activity of TFPI. The term can be used

interchangeably with the term "anti-TFPI antibody". Herein, clone T417 and clone T308

is an antibody are antibodies isolated after immunization of mice with recombinant

human TFPI, and clone 308 is an antibody prepared by humanization of clone T417. In addition, clone 308-2 and clone 308-4 are antibodies prepared by mutating the lysine (K) of the heavy chain of clone 308 with glutamine (Q) or glutamate (E) as shown in FIG. 5.

The equilibrium dissociation constant (KD) of the anti-TFPI antibody may be, for

example, as follows. The KD of clone 308 may be 5.5x10 1 M or lower, preferably

5.25x10-" M or lower, more preferably 5.0x10-1 2 M or lower; the KD of clone 308-2 may

be 3.63x10 1 M or lower, preferably 3.465x10 1 M or lower, more preferably 3.3x10-"

M or lower; and the KD of clone 308-4 may be 2.64x10 1 M or lower, preferably

2.52x10 1 M or lower, more preferably 2.4x10 1 M or lower.

As used herein, the term "clone 1001", "clone 1015", "clone 1021", "clone 1023",

"clone 1024", "clone 1104", "clone 1123", "clone 1202", "clone 1208", "clone 1214",

"clone 1216", "clone 1223", "clone 1224", "clone 1232", "clone 1234", "clone 1238",

"clone 1243", "clone 1248", "clone 3007", "clone 3016", "clone 3024", "clone 3115",

"clone 3120", "clone 3131", "clone 3203", "clone 3241", "clone 4011", "clone 4017",

"clone 4034", "clone 4041", "clone 4141", "clone 4146", "clone 4206", "clone 4208",

"clone 4278", "clone 4287", "clone 1", "clone 2", "clone 3", "clone 4", "clone 5", "clone

6", "clone 7", "clone 8", "clone 9", "clone 10", "clone 11", "clone 12", "clone 13", "clone

14", "clone 15", "clone 16", "clone 17", "clone 18", "clone 19", "clone 20", "clone 21",

"clone 22", "clone 23", "clone A24", "clone A25", "clone A52", "clone A63", "clone

A67", "clone A71" or "clone A74" that binds specifically to TFPI means an antibody that

binds to TFPI, resulting in inhibition of the biological activity of TFPI. The term can be

used interchangeably with the term "anti-TFPI antibody".

In addition, as used herein, the term "clone T417", "clone T308", "clone 308",

"clone 308-2" or "clone 308-4" that binds specifically to TFPI means an antibody that

used interchangeably with the term "anti-TFPI antibody". Herein, clone T417 and clone

T308 is an antibody are antibodies isolated after immunization of mice with recombinant

human TFPI, and clone 308 is an antibody prepared by humanization of clone T417. In

addition, clone 308-2 and clone 308-4 are antibodies prepared by mutating the lysine (K)

of the heavy chain of clone 308 with glutamine (Q) or glutamate (E) as shown in FIG. 5.

example, as follows. The KD of clone 12 may be 9.009X10- 12 M or lower, preferably

8.59x10- 12 M or lower, more preferably 8.19x1O-12 M or lower; the KDof clone 1023 may

be 3.31x10- M or lower, preferably 3.16x10- 1 M or lower, more preferably 3.01x10-"

M or lower; the KDofclone 1202 may be 10.42x10- 12 M or lower, preferably 9.94x10- 12

M or lower, more preferably 9.47x10- 12 M or lower; and the KDof clone 3241 may be

8.14x10-"M or lower, preferably 7.77x10-" M or lower, more preferably 7.4x10-"M or

lower.

In another example of the present invention, the heavy chain variable region and

light chain variable region genes that bind to human TFPI were examined, and then the

heavy chain variable region gene was linked to the human IgG4 heavy chain constant

region, and the light chain variable region gene was linked to the human light-chain

constant region. Next, each of these genes was inserted into a protein expression vector

for an animal cell to thereby construct vectors. The constructed vectors were transfected

into the Expi293 cell line which was then cultured to produce antibodies. The produced

antibodies were purified with protein A (Example 1).

constant region. Next, each of these genes was inserted into a protein expression vector for an animal cell to thereby construct vectors. The constructed vectors were transfected into the Expi293 cell line which was then cultured to produce antibodies. The produced antibodies were purified with protein A (Examples 2 and 3).

Thus, in another aspect, the present invention is directed to a nucleic acid

encoding the antibody. The nucleic acids that are used in the present invention may be

present in a cell lysate, or in a partially purified or substantially pure form. A nucleic

acid is "isolated" or "rendered substantially pure" when purified away from other cellular

components or other contaminants, e.g., other cellular nucleic acids or proteins, by

standard techniques, including alkaline/SDS treatment, CsCl banding, column

chromatography, agarose gel electrophoresis and others well known in the art. The

nucleic acid in the present invention may be, for example, DNA or RNA, and may

comprise or may not comprise an intron sequence.

In still another aspect, the present invention is directed to a vector comprising the

nucleic acid. For expression of an antibody or an antibody fragment thereof, a DNA

encoding a partial or full-length light chain and heavy chain can be obtained by standard

molecular biology techniques (e.g., PCR amplification or cDNA cloning using a

hybridoma that expresses the antibody of interest), and the DNA can be inserted into an

expression vector such that it is operatively linked to transcriptional and translational

control sequences.

As used herein, the term "operatively linked" is intended to mean that an

antibody gene is ligated into a vector such that transcriptional and translational control

sequences serve their intended function of regulating the transcription and translation of

the antibody gene. The expression vector and expression control sequences are chosen to

be compatible with the expression host cell used. An antibody heavy chain gene and an

antibody light chain gene can be inserted into separate vectors, or both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present).

In some cases, the recombinant expression vector can encode a signal peptide that

facilitates secretion of the antibody chain from a host cell. The antibody chain gene can

be cloned into the vector such that the signal peptide is linked in-frame to the amino

terminus of the antibody chain gene. The signal peptide can be an immunoglobulin

signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non

immunoglobulin protein). In addition, the recombinant expression vectors carry

regulatory sequences that control the expression of the antibody chain genes in a host cell.

The term "regulatory sequence" is intended to include promoters, enhancers and other

expression control elements (e.g., polyadenylation signals) that control the transcription

or translation of the antibody chain genes. It will be appreciated by those skilled in the

art that the design of the expression vector, including the selection of regulatory

sequences, may depend on such factors as the choice of the host cell to be transformed,

the level of expression of protein desired, etc.

In yet another aspect, the present invention is directed to a host cell comprising

the nucleic acid or the vector. The nucleic acid or the vector is transfected into a host cell.

Transfection can be performed using various techniques that are generally used to

introduce foreign nucleic acid (DNA or RNA) into procaryotic or eukaryotic cells, for

example, electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection or

lipofection. The antibody according to the present invention can be expressed in

eukaryotic cells, preferably mammalian host cells, in view of its applicability to

mammalian cells. Examples of mammalian host cells suitable for expression of the

antibody include Chinese hamster ovary (CHO) cells (including dhfr-CHO cells that are used together with, for example, a DHFR selectable marker), NSO myeloma cells, COS cells, and SP2 cells.

In yet another aspect, the present invention is directed to a method for producing

an antibody, which comprises culturing a host cell to express the antibody. When a

recombinant expression vector encoding the antibody gene is introduced into mammalian

host cells, the antibody gene can be produced by culturing the host cells for a period of

time such that the antibody is expressed in the host cells, preferably a period of time such

that the antibody is secreted into the medium during culture of the host cells.

In some cases, the expressed antibody can be isolated and purified from the host

cells. Isolation or purification of the antibody can be performed by conventional

isolation/purification methods (e.g., chromatography) that are used for proteins.

Examples of the chromatography include affinity chromatography including a protein A

column and a protein G column, ion exchange chromatography, and hydrophobic

chromatography. In addition to the chromatography, a combination of filtration,

ultrafiltration, salting out, dialysis and the like may be used to isolate and purify the

antibody.

In still another example of the present invention, an FXa activity assay was

performed to evaluate the effects of anti-TFPI antibodies (Example 7). As a result, as

shown in FIG. 9, it was found that absorbance increased in a concentration-dependent

manner in both clone T308 and clone T417 which are chimeric antibodies among anti

TFPI antibody candidates, indicating that the TFPI inhibitory effects of the two

antibodies increase in an antibody concentration-dependent manner. When the effects

were compared at a TFPI concentration of 10 nM, it could be seen that the TFPI

inhibitory activity of clone T417 is better than that of clone T308.

In addition, as shown in FIG. 10, clone 308 was obtained by a humanization

process using clone T417 determined to have a better effect in the above-described assay.

Clone 308 also showed a concentration-dependent increase in absorbance, indicating that

it could inhibit TFPI.

Furthermore, as shown in FIG. 11, back mutation was performed in order to

increase the effect of clone 308, and clone 308-2 and clone 308-4 were obtained. It could

be seen that both clone 308-2 and clone 308-4 inhibited TFPI in a concentration

dependent manner. Also, when samples treated with 40 nM and 10 nM were compared,

it could be seen that the TFPI inhibitory activities of clone 308-2 and clone 308-4

increased compared to that of clone 308. At a concentration of 40 nM, clone 308-2 and

clone 308-4 showed TFPI inhibitory activities of 85% and 82%, respectively, compared

to a positive control (mAb202l or anti-TFPI Ab), but at a concentration of 10 nM, clone

308-2 showed a TFPI inhibitory activity of 72%, and clone 308-4 showed a TFPI

inhibitory activity of 78%, which was better than that of clone 308-2. In addition, it was

found that the clone antibodies showed TFPI inhibitory activities equal to that of clone

T417 chimeric antibody showing a TFPI inhibitory activity of 77%.

In still another example of the present invention, a TF/FVIIa/FXa complex assay

was performed to evaluate the effects of anti-TFPI antibodies (Example 8). Specifically,

in a state in which TFPI was present together with or independently of anti-TFPI

antibodies, the extents of production and inhibition of FXa by a TF/FVIIa complex were

evaluated based on FXa activity.

As a result, as shown in FIG. 12, clone T308 and clone T417 antibodies that are

chimeric antibodies among anti-TFPI antibody candidates showed a concentration

dependent increase in absorbance, indicating that the TFPI inhibitory effects of the two antibodies increase in a concentration-dependent manner. Particularly, it could be seen that the TFPI inhibitory activity of clone T417 was better than that of clone T308.

In addition, as shown in FIG. 13, clone 308 was obtained by a humanization

process using clone T417 antibody having a better effect than clone T308. It could be

seen that clone 308 also showed a concentration-dependent increase in absorbance,

indicating that it inhibits TFPI.

Furthermore, as shown in FIG. 14, back mutation was performed in order to

increase the effect of clone 308 humanized antibody. As a result, the TFPI inhibitory

activity of clone 308-2 or clone 308-4 increased compared to that of clone 308. At a

concentration of 25 nM, clone 308-2 showed a TFPI inhibitory activity of 37.8%, and

clone 308-4 showed a TFPI inhibitory activity of 68.4%, which was higher than that of

clone 308-2.

In still another example of the present invention, a thrombin generation assay for

clone 308-2 and clone 308-4, selected through the FXa activity assay and the

TF/FVIIa/FXa complex assay, was performed (Example 9). As a result, as shown in FIG.

15, both clone T417 and clone 308 showed increases in the thrombin generation peak and

the thrombin generation compared to a negative control group (having no antibody). In

samples treated with 2.5 nM, clone T417 and clone 308 showed thrombin peak values of

208% and 162%, respectively, compared to a negative control group (having no

antibody), and the ETP values indicating thrombin generation were 131% in clone T417

and 122% in clone 308. Thus, it was found that clone T417 has a better effect than clone

308 antibody.

Moreover, as shown in FIG. 16, clone 308-2 and clone 308-4 showed increases

in the thrombin generation peak and the total thrombin generation compared to clone 308

antibody. In particular, in samples treated with 2.5 nM, both clone 308-2 and clone 308

4 showed increases in thrombin peak value of 183% and 191%, respectively, compared to

a negative control group (having no antibody), and the ETP value was 126% in both

clone 308-2 and clone 308-4, indicating that the clone antibodies have an increased

ability to produce thrombin.

In another example of the present invention, an FXa activity assay was

performed to evaluate the effects of anti-TFPI antibodies (Example 5). As a result, as

shown in FIGS. 20 to 28, the effects of affinity-matured antibodies among anti-TFPI

antibody candidates were demonstrated. It was found that the antibodies showed

increases in the absorbance in an antibody concentration-dependent manner, indicating

that the TFPI inhibitory effect of the antibodies increases in an antibody concentration

dependent manner.

In another example of the present invention, a TF/FVIIa/FXa complex assay was

performed to evaluate the effects of anti-TFPI antibodies (Example 15). Specifically, the

extents of production and inhibition of FXa by a TF/FVIIa complex were evaluated based

on FXa activity in a state in which TFPI were present together with or independently of

anti-TFPI antibodies. As a result, as shown in FIGS. 29 to 33, the effects of affinity

matured antibodies among anti-TFPI antibody MG1113 candidates were demonstrated. It

was found that the antibodies showed increases in the absorbance in an antibody

concentration-dependent manner, indicating that the TFPI inhibitory effect of the

candidate antibodies increases in an antibody concentration-dependent manner.

anti-TFPI antibodies selected through the FXa activity assay was performed (Example

16). As a result, as shown in FIG. 34, for No. 1023 antibody among affinity-matured

antibody candidates selected through the Fxa activity assay and the TF/FVIIa/FXa

complex assay, a thrombin generation comparison assay was performed using T417 chimeric antibody. At 2.5 nM, T417 antibody showed an increase in thrombin peak of about 335% compared to a blank treated with only a sample dilution, and No. 1023 antibody showed an increase in thrombin peak of about 401% compared with the blank.

In addition, in the case of ETP indicating the total generation of thrombin, T417 antibody

showed an increase in ETP of about 293% compared to a negative control group (having

no antibody) at a concentration of 2.5 nM, and No. 1023 antibody showed an increase in

ETP of about 309% compared to the negative control group. The comparison between

the two antibodies indicated that No. 1023 antibody obtained from affinity maturation has

a better effect than the T417 antibody.

In a further aspect, the present invention is directed to a pharmaceutical

composition for treating hemophilia, which comprises an anti-TFPI antibody as an active

ingredient.

The present invention also provides a pharmaceutical composition comprising a

therapeutically effective amount of an anti-TFPI antibody and a pharmaceutically

acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a substance

which can be added to the active ingredient to help formulate or stabilize the preparation

and causes no significant adverse toxicological effects to the patient.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier

or diluent that does not impair the biological activity and characteristics of an

administered compound without irritating an organism. As a pharmaceutically

acceptable carrier in a composition that is formulated as a liquid solution, a sterile and

biocompatible carrier is used. The pharmaceutically acceptable carrier may be

physiological saline, sterile water, Ringer's solution, buffered saline, albumin injection

solution, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture of two

or more thereof. In addition, the composition of the present invention may, if necessary, comprise other conventional additives, including antioxidants, buffers, and bacteriostatic agents. Further, the composition of the present invention may be formulated as injectable forms such as aqueous solutions, suspensions or emulsions with the aid of diluents, dispersants, surfactants, binders and lubricants. In addition, the composition according to the present invention may be formulated in the form of pills, capsules, granules, or tablets.

Other carriers are described in a literature [Remington's Pharmaceutical Sciences (E. W.

Martin)]. This composition may contain a therapeutically effective amount of at least one

anti-TFPI antibody.

Pharmaceutically acceptable carriers include sterile aqueous solutions or

dispersions and sterile powders for the extemporaneous preparation of sterile injectable

solutions or dispersions. The use of such media and agents for pharmaceutically active

substances is known in the art. The composition is preferably formulated for parenteral

injection. The composition can be formulated as a solid, a solution, a microemulsion, a

liposome, or other ordered structures suitable to high drug concentration. The carrier

may be a solvent or dispersion medium containing, for example, water, ethanol, polyol

(e.g., glycerol, propylene glycol and liquid polyethylene glycol), and suitable mixtures

thereof. In some cases, the composition may contain an isotonic agent, for example,

sugar, polyalcohol, sorbitol or sodium chloride. Sterile injectable solutions can be

prepared by the active compound in the required amount in an appropriate solvent with

one or a combination of ingredients enumerated above, as required, followed by filtered

sterilization. Generally, dispersions are prepared by incorporating the active compound

into a sterile vehicle, which contains a basic dispersion medium and the required other

ingredients from those enumerated above. In the case of sterile powders for the

preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Anti-TFPI antibodies can be used for therapeutic purposes for treating genetic

and acquired deficiencies or defects in coagulation.. For example, the antibodies can be

used to block the interaction between TFPI and FXa, or to prevent the TFPI-dependent

inhibition of TF/FVIla activity. Additionally, the human monoclonal antibody may also

be used to restore the TF/FVIla-driven generation of FXa to bypass the insufficiency of

FVIII- or FIX-dependent amplification of FXa.

The antibodies have therapeutic use in the treatment of disorders of hemostasis

such as thrombocytopenia, platelet disorders and bleeding disorders (e.g., hemophilia A

and hemophilia B). Such disorders may be treated by administering a therapeutically

effective amount of the anti-TFPI antibody to a patient in need thereof. The antibodies

also have therapeutic use in the treatment of uncontrolled bleeds in indications such as

trauma and hemorrhagic stroke. Thus, the present invention also provides a method for

shortening the bleeding time comprising administering a therapeutically effective amount

of the anti-TFPI antibody to a patient in need thereof.

The antibody can be used as monotherapy or in combination with other therapies

to address a hemostatic disorder. For example, co-administration of one or more

antibodies of the present invention with a clotting factor such as TF (tissue factor), FVII

(factor VII) or FX (factor X) is believed useful for treating hemophilia. By co

administration or combination therapy of the antibody with a clotting factor is meant

administration of the two therapeutic drugs each formulated separately or formulated

together in one composition, and, when formulated separately, administered either at

approximately the same time or at different times, but over the same therapeutic period.

The pharmaceutical compositions may be parenterally administered to subjects

suffering from hemophilia A or B at a dosage and frequency that may vary with the

severity of the bleeding episode or, in the case of prophylactic therapy, may vary with the

severity of the patient's clotting deficiency. The compositions may be administered to

patients in need as a bolus or by continuous infusion. For example, a bolus

administration of the inventive antibody present as a Fab fragment may be in an amount

of from 0.0025 to 100 mg/kg body weight, 0.025 to 0.25 mg/kg, 0.010 to 0.10 mg/kg or

0.10-0.50 mg/kg. For continuous infusion, the inventive antibody present as an Fab

fragment may be administered at 0.001 to 100 mg/kg body weight/minute, 0.0125 to 1.25

mg/kg/min, 0.010 to 0.75 mg/kg/min, 0.010 to 1.0 mg/kg/min or 0.10-0.50 mg/kg/min for

a period of 1-24 hours, 1-12 hours, 2-12 hours, 6-12 hours, 2-8 hours, or 1-2 hours. For

administration of the inventive antibody present as a full-length antibody (with full

constant regions), dosage amounts may be about 1-10 mg/kg body weight, 2-8 mg/kg, or

5-6 mg/kg. Such full-length antibodies would typically be administered by infusion

extending for a period of 30 minutes to 35 minutes. The frequency of the administration

would depend upon the severity of the condition. Frequency could range from three

times per week to once every one week or two weeks.

Additionally, the compositions may be administered to patients via subcutaneous

injection. For example, a dose of 10 to 100 mg anti-TFPI antibody can be administered

to patients via subcutaneous injection weekly, biweekly or monthly.

As used herein, "therapeutically effective amount" means an amount of an anti

TFPI antibody variant or of a combination of such antibody and TF (tissue factor), FVII

(factor VII) or FX (factor X) that is needed to effectively increase the clotting time in

vivo or otherwise cause a measurable benefit in vivo to a patient in need. The precise

amount will depend upon numerous factors, including, but not limited to the components and physical characteristics of the therapeutic composition, intended patient population, individual patient considerations, and the like, and can readily be determined by one skilled in the art. When these factors are completely considered, it is important to administer the minimum amount sufficient for achieving the highest effect without causing side effects, and this dose can be easily determined by those skilled in the art.

The dose of the pharmaceutical composition of the present invention may vary

depending on various factors, including a patient's health condition and weight, severity

of a disease, the type of drug, and the route and period of administration. The

composition may be administered in a single dose or in multiple doses per day into

mammals including rats, mice, domestic animals, humans, etc. via any typically accepted

route, for example, orally, rectally, intravenously, subcutaneously, intrauterinely, or

intracerebrovascularly.

EXAMPLES

Hereinafter, the present invention will be described in further detail with reference

to examples. It will be obvious to a person having ordinary skill in the art that these

examples are illustrative purposes only and are not to be construed to limit the scope of

the present invention.

Example 1: Preparation of Anti-TFPI Antibody

As an antibody against TFPI (tissue factor pathway inhibitor) that inhibits the

factor X activity, an antibody for treating or preventing hemophilia, which can prevent

the inhibition of blood coagulation, was prepared.

1-1: Selection of Antibody

Mice were immunized with recombinant human TFPI, and the spleens were

extracted from the mice. B lymphocytes were extracted from the spleens, total RNA was isolated therefrom, and then synthesized into cDNA. From the synthesized cDNA, various mouse antibody genes were cloned by PCR (polymerase chain reaction), and inserted into pComb3X phagemids, thereby constructing an antibody library displaying antibody fragments having various sequences. In order to select a human TFPI-specific antibody from the antibody library, TFPI-immobilized magnetic beads and the antibody library were mixed with each other, and clones binding to the target antigen were separated and cultured. Then, clones (T417 or T308 clone cells) binding specifically to the target antigen (human TFPI) were individually selected by ELISA (enzyme linked immunosorbent assay), and the amino acid sequences of the antibody genes were identified by sequencing.

As a result, as shown in Table 1 below, clone T417 and clone T308, which bind

specifically to human TFPI, could be selected, and the amino acid sequences thereof were

identified.

Table 2 below the CDR amino acid sequences of the clone antibodies of Table 1,

identified based on the Kabat numbering system.

Table 1 Clones Variable AA Sequences SEQ ID

Regions NOS:

T417 VH EVHLVESGGDLVKPGGSLKLSCAASGFTFSSY 1 AMSWVRQTPDKRLEWVATITTGGSYTYYPDS VKGRFTISRDNAKNTLYLQMSSLKSEDTAMY YCARQDGNFLMDYWGQGTTVTVSS VL DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSD 2 GKTYLNWLLQRPGQSPKRLIYLVSKLDSGVP DRFTGSGSGTDFTLKISRVEAEDLGVYYCWQ GTHFPFTFGSGTKLEIKR T308 VH EVKLVESGGGLVKPGGSLKLSCAASGFTFSNY 3 PMSWVRQTPEKRLEWVATISNSGSYTYYPDS VKGRFTISRDNAKNTLYLQMNSLRSEDTAMY YCARQVYGNYEDFDYWGQGTTLTVSS

VL DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSD 4 GKTYLNWLLQRPGQSPKRLIYLVSKLDSGVP DRFTGSGSGTDFTLKISRVEAEDLGVYYCWQ GTHFPYTFGGGTKLELKR

Table 2 Clones Variable Regions CDR1 CDR2 CDR3

SYAMS TITTGGSYTYY QDGNFLMDY

Heavy Chain (SEQ ID NO: 5) PDSVKG (SEQ ID NO: 7)

(SEQ ID NO: 6) T417 KSSQSLLDSDG LVSKLDS WQGTHFPF

Light Chain KTYLN (SEQ ID NO: 9) (SEQ ID NO:

(SEQ ID NO: 8) 10)

NYPMS TISNSGSYTYY QVYGNYEDFD

(SEQ ID NO: PDSVKG Y Heavy Chain 11) (SEQ ID NO: (SEQ ID NO:

12) 13) T308 KSSQSLLDSDG LVSKLDS WQGTHFPY

KTYLN (SEQ ID NO: (SEQ ID NO: Light Chain (SEQ ID NO: 15) 16)

14)

1-2: Cloning of IgG Genes of T417 and T308 Clone Antibodies

From the T417 and T308 clone cells, pComb3X phagemids containing the genes

encoding the heavy-chain variable regions of the T417 and T308 clone antibodies were extracted. Using each of the extracted pComb3X phagemids as a template, PCR was performed using Accupower Pfu PCR premix (Bioneer) together with an NotI-containing forward primer (Table 3; SEQ ID NO: 17) and an ApaI-containing reverse primer (Table

3; SEQ ID NO: 18). The PCR was performed under the following conditions: 10 min at

94°C; and then 30 cycles, each consisting of 15 see at 94°C, 30 see at 56°C and 90 see at

72°C; followed by 10 min at 72°C. The amplified genes were electrophoresed on 1%

agarose gel to confirm the DNA bands having the expected sizes, and were isolated using

a gel extraction kit. Next, each of the isolated genes was treated with NotI and Apal

restriction enzymes at 37°C for 12 hours or more. The gene treated with the restriction

enzyme was separated on 1% agarose gel. A pcIW plasmid vector containing the IgG4

heavy chain constant region gene was also digested in the same manner and separated on

agarose gel. Using T4 DNA ligase (Cat.No.M0203S, New England BioLabs(NEB)),

each of the isolated T417 and T308 heavy-chain variable region genes was ligated into

the KpnI and Apal sites of a linear pcw vector containing the human heavy-chain

constant region. The ligation product was transformed into XL1-Blue bacteria

(Electroporation-Competent Cells; Cat.No.200228, Stratagene), and the bacterial cells

were plated on a carbenicillin-containing LB plate (Cat.No.LN004CA, NaraeBiotech),

and then cultured at 37°C for 12 hours or more. Next, single colonies were selected from

the plate and cultured, and a plasmid was separated therefrom using a plasmid mini-kit

(Cat.No.27405, QIAGEN) and identified by DNA sequencing.

From the T417 and T308 clone cells, pComb3X phagemids containing the genes

encoding the light-chain variable regions of the T417 and T308 clone antibodies were

extracted. Using each of the extracted pComb3X phagemids as a template, PCR was

performed using Accupower Pfu PCR premix together with an NotI-containing forward

primer (Table 3; SEQ ID NO: 19) and a KpnI-containing reverse primer (Table 3; SEQ

ID NO: 20). The PCR was performed under the following conditions: 10 min at 94°C;

and then 30 cycles, each consisting of 15 see at 94°C, 30 see at 56°C and 90 see at 72°C;

followed by 10 min at 72°C. The amplified genes were electrophoresed on 1% agarose

gel to confirm the DNA bands having the expected sizes, and were isolated using a gel

extraction kit. Next, each of the isolated genes was treated with NotI and KpnI restriction

enzymes at 37°C for 12 hours or more. The gene treated with the restriction enzyme was

separated on 1% agarose gel. A pcIW plasmid vector was also digested in the same

manner and separated on agarose gel. Using T4 DNA ligase (Cat.No.M0203S, New

England BioLabs(NEB)), each of the isolated T417 and T308 light-chain variable region

genes was ligated into the NotI and KpnI sites of a linear pcw vector containing the

human light-chain constant region. The ligation product was transformed into XL1-Blue

bacteria (Electroporation-Competent Cells; Cat.No.200228, Stratagene), and the bacterial

cells were plated on a carbenicillin-containing LB plate (Cat.No.LN004CA,

NaraeBiotech), and then cultured at 37°C for 12 hours or more. Next, single colonies

were selected from the plate and cultured, and a plasmid was separated therefrom using a

plasmid mini-kit (Cat.No.27405, QIAGEN) and identified by DNA sequencing.

Table 3

Names DNA Sequences SEQ ID NOS: GCGGCCGCCATGTATCTG GGTCTGAACTATGTCTTT T417VH-F ATCGTGTTTCTGCTGAAT 17 GGTGTGCAGTCTGAGGTG CACCTGGTGGAGTCT NNNNGGGCCCCTTGGTG T417VH Apa-R CTGGCTGAGGAGACGGT 18 GACCGTGGT

GCGGCCGCCATGGATAG CCAGGCTCAGGTGCTGAT GCTGCTGCTGCTGTGGGT T417 VL-F 19 GTCAGGGACTTGCGGGG ACGTTGTGATGACCCAGA CTCCACT NNNNGGTACCAGATTTC VL-R 20 AACTGCTCATCAGA

1-3: Production and Purification of Anti-TFPI T417, T308 Clone Antibody Mutant IgG

In order to produce and purify the anti-TFPI clone T417 and T308 clones

obtained by mouse immunization, Expi293FTM cells were seeded at a concentration of 2.5

X 106 cells/mL on one day before transfection. After 24 hours of culture (37°C, 8% C02,

125 rpm), Expi293 T M Expression medium (Cat.No.A1435101, Gibco) was added to

prepare 30 mL of the cells at a concentration of 2.5 X 106 cells/mL (viability > 95%). 30

g of DNA (pcw-anti-TFPI heavy chain: 15[g, pclw-anti-TFPI light chain: 15kg) was

diluted in 1.5 mL of OptiProTMSEM medium (Cat.No.12309019, Gibco) to a total

volume of 1.5 mL and incubated at room temperature for 5 minutes. 80 L of

ExpiFectamineTM293 reagent (Cat.No.A14524, Gibco) was added to 1.5 mL of

OptiProTMSEM medium (Cat.No.12309019, Gibco) to a total volume of 1.5 mL, and

then incubated at room temperature for 5 minutes. After 5 minutes of incubation, 1.5 mL

of the diluted DNA and 1.5 mL of the ExpiFectamine TM 293 reagent were mixed well

with each other and incubated at room temperature for 20-30 minutes. Expi293FTM cells

were treated with 3 mL of the mixture of the DNA and the ExpiFectamine TM 293reagent.

After 16-18 hours of suspension culture (37°C, 8% C02, 125 rpm), 150 L of

ExpiFectamine TM 293 Enhancer 1 (Cat.No.A14524, Gibco) and 1.5 mL of

ExpiFectamine T M 293 Enhancer 2 (Cat.No.A14524, Gibco) were added to the cells,

followed by suspension culture for 5 days. After completion of the culture, the cells were

centrifuged at 4000 rpm for 20 minutes to remove cell debris, and the supernatant was

passed through a 0.22 m filter. 100 L of the protein A resin MabSelect Xtra

(Cat.No.17-5269-02, GE Healthcare) was prepared per 30 mL of the culture medium,

centrifuged at 1000 rpm for 2 minutes to remove the storage solution, and washed three

times with 400 L of protein A binding buffer (Cat.No.21007, Pierce) for each washing.

Protein A resin was added to the prepared culture medium, followed by rotating

incubation at room temperature for 30 minutes. The mixture of the culture medium and

the resin was added to the Pierce spin column-snap cap (Cat.No.69725, Thermo), and

extracted using the QIAvac 24 Plus (Cat.No.19413, QIAGEN) vacuum manifold so that

only the resin remained in the column. The resin was washed with 5 mL of protein A

binding buffer, and then resuspended in 200 IL of protein A elution buffer

(Cat.No.21009, Pierce), after which it was incubated at room temperature for 2 minutes

and eluted by centrifugation at 1000 rpm for 1 minute. The eluate was neutralized by

addition of 2.5 L of 1.5M Tris-HCl (pH 9.0). Elution was performed 4-6 times, and

each fraction was quantified using Nanodrop 200C (Thermo Scientific). Fractions having

the protein detected therein were collected, and the buffer was replaced with PBS

(phosphate-buffered saline) buffer using 5 mL of 7K MWCO (Cat.No.0089892, Pierce)

in Zeba Spin Desalting Columns. Next, electrophoresis (SDS-PAGE) of the protein was

performed under reducing and non-reducing conditions to finally quantify the

concentration of the antibody and verify the state of the antibody, and the antibody was

stored at 4°C.

As a result, as shown in FIG. 3, protein electrophoresis (SDS-PAGE) indicated

that the T417 and T308 clone antibodies were purified in a good state.

Example 2: Construction of Humanized Antibody by CDR-Grafting to Stable

Framework

When the quantitative binding affinities of the TFPI antigen (full-length human

TFPI protein) (Cat.No.TFPI-875H; Creative Biomart, USA) for the T417 and T308 clone

antibodies were evaluated, the clone T417 antibody showed the best effect (see FIG. 8

and Example 6). Thus, humanization of clone T417 was performed in order to clone 308.

In order to humanize the mouse-derived clone T417 antibody, CDR-grafting that

is most widely used for humanization was selected. Specifically, the structure of clone

T417 was predicted through a sample showing the highest QMEAN, GMQE and

homology values among 50 samples obtained from Swiss-Model

(http://swissmodel.expasy.org/) that is a structure prediction site, and the CDRs binding

to the antigen and a framework other than the CDRs were identified using the Kabat and

Chothia numbering scheme. Then, a human framework having the highest homology

was searched using IgBLAST (http://www.ncbi.nlm.nih.gov/igblast/). From several

combinations of several heavy-chain variable regions and light-chain variable regions

obtained by the search, VH3-21/VK2-30 showing the highest formation rate in human

germline cell analysis was selected (de Wildt RM et al., J Mol. Biol., 285:895-901, 1999;

mAbs, 5:3, 445-470). Next, clone 308 that is a humanized antibody of clone T417 was

constructed, which comprises: the light-chain variable region K24 of clone T417, which

is a framework sequence but does not influence the antibody stability, and is also present

in the human antibody sequence; and the heavy-chain variable region N35 which is a

CDR sequence identified based on the Kabat numbering system, but is a framework

sequence in structural terms (Methods, 34:184-199, 2004; http://www.vbase2.org/) (see

FIG. 4 and Table 4 below).

As a result, as shown in FIG. 4, clone 308 was constructed by humanization of

clone T417.

Table 5 below the CDR amino acid sequences of the clone antibody of Table 4,

identified based on the Kabat numbering system.

Table 4 Clones Variable AA Sequences SEQ ID

Regions NOS:

308 VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSSY 21 AMNWVRQAPGKGLEWVSTITTGGSYTYYAD SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY YCARQDGNFLMDYWGQGTLVTVSS VL DVVMTQTPLSLPVTLGQPASISCKSSQSLLDSD 22 GKTYLNWLQQRPGQSPKRLIYLVSKLDSGVP DRFTGSGSGTDFTLKISRVEAEDVGVYYCWQ GTHFPFTFGQGTKVEIKR

Table 5 clones Variable CDR1 CDR2 CDR3 Regions

308 Heavy SYAMN TITTGGSYTYYPDSVKG QDGNFLMDY

Chain (SEQ ID NO:23) (SEQ ID NO:6) (SEQ ID NO:7)

Light KSSQSLLDSDGKTYLN LVSKLDS WQGTHFPF

Chain (SEQ ID NO:8) (SEQ ID NO:9) (SEQ ID

NO:10)

Example 3: Design of Clone 308 Antibody Mutant by in silico Modeling

The binding between clone 308 constructed in Example 2 and TFPI KPI-2

(Kunitz domain 2) was predicted by in silico modeling, and a position that can improve

the binding to the antigen was predicted (Heavy chain-52a, -64 and light chain 27d) (see

FIG. 5 and Table 6 below).

Using homology modeling that is the BioLuminate module (Schrodinger, USA),

the structure of the clone 308 antibody that binds to TFPI was produced. To produce the structure, a template search was performed through the PDB database using the sequence of clone 308. As a result, a 3QOS (PDB number) structure having a similar structure and a high composite score was selected. It could be seen that 3QOS and clone 308 are similar in sequences other than the HV CDR H3 region having an antigen-specific structure and are templates suitable for producing the structure. A total of clone 308 models were produced and compared with the structure of 3QOS, and the most similar structure was finally selected. The selected model was similar in structures other than the

HV CDR H3 region, and the interaction between clone 308 and the TFPI structure was

predicted using the protein-protein binding prediction program PIPER (see FIG. 17 in

which the molecule indicated by green indicates the 308 clone antibody and the molecule

indicated by red indicates the TFPI antigen). Thus, the predicted paratope of the clone

308 antibody and the predicted epitope of the human TFPI antibody that binds thereto

could be identified (Table 8). Based on the predicted binding structure, a mutation was

introduced into the amino acid sequence of clone 308 in order to increase the affinity of

clone 308. In other words, K64 was replaced with Q and E so as to induce an ionic bond

with R17 of TFPI.

As a result, as shown in Table 6 below, DNA sequencing indicated that a total of

two clone 308 mutants were constructed. The predicted binding between the heavy-chain

variable region or light-chain variable region of clone 308 and the human TFPI antigen is

shown in FIGS. 18 and 19.

Table 7 below shows the CDR amino acid sequences of the clone antibodies of

Table 6, identified based on the Kabat numbering system.

Table 8 below shows the predicted paratope of the clone 308 antibody and the

predicted epitope of the human TFPI antigen that binds thereto.

Table 6 Clones Variable Regions AA Sequences SEQ ID NOS: EVQLVESGGGLVK PGGSLRLSCAASGF TFSSYAMNWVRQA PGKGLEWVSTITTG 308-2 Heavy Chain GSYTYYADSVQGR 24 FTISRDNAKNSLYL QMNSLRAEDTAVY YCARQDGNFLMD YWGQGTLVTVSS EVQLVESGGGLVK PGGSLRLSCAASGF TFSSYAMNWVRQA PGKGLEWVSTITTG 308-4 Heavy Chain GSYTYYADSVEGR 25 FTISRDNAKNSLYL QMNSLRAEDTAVY YCARQDGNFLMD YWGQGTLVTVSS

Table 7 clones Variable CDR1 CDR2 CDR3 Regions 308-2 Heavy SYAMN TITTGGSYTYYPDSVQG QDGNFLMDY Chain (SEQ ID (SEQ ID NO: (SEQ ID NO: 7) NO: 26) 23) 308-4 Heavy SYAMN TITTGGSYTYYPDSVEG QDGNFLMDY Chain (SEQ ID (SEQ ID NO: 27) (SEQ ID NO: 7) NO: 23)

Table 8

Paratope of Clone 308 Epitope of hTFPI Type of binding HCDR1 SL1 Q29 Hydrogen bond HCDR2 T52 E11 Hydrogen bond HCDR2 T52a E10 Hydrogen bond HCDR2 Y56 P13 Hydrophobic iriteaction HCDR2 Y8 K Hydrogen bond HCDR2 Y59 RI7 Hydrogen bond HICDR2 D61 R27 Sak bridge HCDR3 Q98 R34 Hydrogen bond LCDRI DM0a Y23 Hydrogen bond LCDR 3 G9 R34 Hydrogen bond LCDR 3 T92 721 Hydrogen bond LCDRT792 R?4 Hydrogen bond LCDR2 T92 K36 Hydrogen bond LCDR B F94 Y19 Hydrophobic interaction

Example 4: Preparation of Clone 308 Antibody Mutant

4-1: Cloning of IgG Gene of Clone 308 Antibody Mutant

Using each of the synthesized 308-2 and 308-4 genes (Bioneer, Korea) as a

template, the heavy-chain variable region was subjected to PCR using PrimeSTAR HS

DNA polymerase (Cat.No.ROB; Takara) together with a KpnI-containing forward

primer (Table 9; SEQ ID NO: 28) and an ApaI-containing reverse primer (Table 9; SEQ

ID NO: 29). The PCR was performed under the following conditions: 2 min at 98°C; and

then 30 cycles, each consisting of 10 see at 98°C, 10 see at 58°C and 30 see at 72°C;

followed by 5 min at 72°C. The amplified gene was electrophoresed on 1% agarose gel

to confirm the DNA band having the expected size, and was isolated using a gel

extraction kit (Cat.No.287041, QIAGEN). The isolated gene was treated with KpnI and

Apal enzyme at 37°C for 4 hours, and then separated on 1% agarose gel. A pcIW plasmid vector was also digested in the same manner and separated on agarose gel.

Using T4 DNA ligase (Cat.No.M0203S, NEB), the isolated gene was ligated into the

KpnI and Apal of a linear pcIW vector. The ligation product was transformed into XL1

Blue bacteria (Electroporation-Competent Cells; Cat.No.200228, Stratagene), and the

bacterial cells were plated on a carbenicillin-containing LB plate (Cat.No.LN004CA,

NaraeBiotech) and cultured at 37°C for 12 hours or more, and single colonies were

selected from the plate and cultured. Next, a plasmid was isolated from the cells using a

plasmid mini-kit (Cat.No.27405, QIAGEN) and identified by DNA sequencing.

Table 9 Names DNA Sequences SEQ ID NOS:

VH Fo TGCTGTGGGTGAGTGGTA 28 CCTGTGGGGAAGTGCAG CTCGTGGAGAGCGGT VH Re AGTGGGAACACGGAGGG 29 CCCCTTGGTGCTGGCGGA TGAGACAGTCACAAGTG TCCC

4-2: Production and Purification of Clone 308 Antibody Mutant IgG

In order to produce and purify 308-2 and 308-4 clones that are clone 308-2 and

308-4 antibody mutants, Expi293FTMcells were seeded at a concentration of 2.5 X 106

cells/mL on one day before transfection. After 24 hours of culture (37°C, 8%CO2, 125

rpm), Expi293 TM Expression medium (Cat.No.A1435101, Gibco) was added to prepare

30 mL of the cells at a concentration of 2.5 X 106 cells/mL (viability > 95%). 30 g of

DNA (pclw-anti-TFPI heavy chain: 15[g, pclw-anti-TFPI light chain: 15kg) was diluted

in OptiProTMSEM medium (Cat.No.12309019, Gibco) to a total volume of 1.5 mL and

incubated at room temperature for 5 minutes. 80 L of ExpiFectamineTM293 reagent

(Cat.No.A14524, Gibco) was added to 1.5 mL of OptiProTMSEM medium

(Cat.No.12309019, Gibco) to a total volume of 1.5 mL, and then incubated at room

temperature for 5 minutes. After 5 minutes of incubation, 1.5 mL of the diluted DNA

and 1.5 mL of the ExpiFectamine T M 293 reagent were mixed well with each other and

incubated at room temperature for 20-30 minutes. Expi293FTM cells were treated with 3

mL of the mixture of the DNA and the ExpiFectamine TM 293 reagent. After 16-18 hours

of suspension culture (37°C, 8% C02, 125 rpm), 150 L of ExpiFectamine TM 293

Enhancer 1 (Cat.No.A14524, Gibco) and 1.5 mL of ExpiFectamine TM 293 Enhancer 2

(Cat.No.A14524, Gibco) were added to the cells, followed by suspension culture for 5

days. After completion of the culture, the cells were centrifuged at 4000 rpm for 20

minutes to remove cell debris, and the supernatant was passed through a 0.22 m filter.

100 L of the protein A resin MabSelect Xtra (Cat.No.17-5269-02, GE Healthcare) was

prepared per 30 mL of the culture medium, centrifuged at 1000 rpm for 2 minutes to

remove the storage solution, and washed three times with 400 L of protein A binding

buffer (Cat.No.21007, Pierce) for each washing. Protein A resin was added to the

prepared culture medium, followed by rotating incubation at room temperature for 30

minutes. The mixture of the culture medium and the resin was added to the Pierce spin

column-snap cap (Cat.No.69725, Thermo), and extracted using the QIAvac 24 Plus

(Cat.No.19413, QIAGEN) vacuum manifold so that only the resin remained in the

column. The resin was washed with 5 mL of protein A binding buffer, and then

resuspended in 200 L of protein A elution buffer (Cat.No.21009, Pierce), after which it

was incubated at room temperature for 2 minutes and eluted by centrifugation at 1000

rpm for 1 minute. The eluate was neutralized by addition of 2.5 L of 1.5M Tris-HCl

(pH 9.0). Elution was performed 4-6 times, and each fraction was quantified using

Nanodrop 200C (Thermo Scientific). Fractions having the protein detected therein were collected, and the buffer was replaced with PBS (phosphate-buffered saline) buffer using

5 mL of 7K MWCO (Cat.No.0089892, Pierce) in Zeba Spin Desalting Columns. Next,

electrophoresis (SDS-PAGE) of the protein was performed under reducing and non

reducing conditions to finally quantify the concentration of the antibody and verify the

state of the antibody, and the antibody was stored at 4°C.

As a result, a total of 18 clone antibodies were prepared by introducing one or

more mutations into four positions of the amino acid sequence of clone 308, which can

enhance the binding of the antibody to the antigen (human recombinant TFPI protein and

were selected based on the prediction of Example 3 (FIG. 5 and Tables 4 to 7; the amino

acid sequences of the 308, 308-2 and 308-4 clone antibodies). Protein electrophoresis

(SDS-PAGE) indicated that the antibodies were purified in a good state (FIG. 6). Among

these antibodies, clone 308-2 and clone 308-4 have a glutamine (Q) or glutamate (E)

mutation introduced into the heavy-chain lysine (K) of clone 308.

Tables 4 and 6 show the heavy-chain and light-chain amino acid sequences of the

anti-TFPI clone antibodies.

Tables 5 and 7 show the CDR amino acid sequences of the clone antibodies of

Tables 4 and 6, identified based on the Kabat numbering system.

Example 5: Preparation of TFPI KPI-2

5-1: Cloning of Human TFPI KPI-2 (Kunitz Domain 2), Rabbit KPI-2 and Mouse TFPI

KPI-2 Genes

In order to construct human TFPI KPI-2 (Kunitz domain 2), rabbit TFPI KPI-2

and mouse TFPI KPI-2 genes (see Table 10), the restriction enzyme sites NcoI

(Cat.No.R0193S, NEB) and NotI (Cat.No.R0189S, NEB) were introduced into pET22b

plasmid vectors. Each gene (synthesized by GeneScript) was subjected to PCR using an

NcoI-containing forward primer (Table 11; SEQ ID NOs: 33 to 35) and an NotI containing reverse primer (Table 11; SEQ ID NOs: 36 to 38). The PCR was performed under the following conditions: 2 min at 94°C; and then 30 cycles, each consisting of 30 see at 94°C, 30 see at 55°C and 30 see at 72°C; followed by 5 min at 72°C. The amplified genes were electrophoresed on 1% agarose gel to confirm the DNA bands having the expected sizes, and were isolated using a gel extraction kit (Cat.No.28704,

QIAGEN). The three isolated genes were treated with NcoI and NotI restriction enzymes at 37°C for 4 hours. The treated genes were separated on 1% agarose gel. A pET22b

plasmid vector was also digested with NcoI and NotI in the same manner and separated

on agarose gel. The prepared pET22b NcoI/NotI vector and the insert were mixed at a

molar ratio of 1:3, and then T4 DNA ligase (Cat.No.M0202S; NEB) and ligase buffer

(Cat.No.B0202S; N EB) were added thereto, followed by incubation at 25°C for 3 hours.

5 pL of the ligation product was added to DH5a (chemical competent cells; Invitrogen)

and incubated on ice for 10 minutes. For heat shock, the cells were incubated at 42°C for

1 minute, and for cell recovery, the cells were suspension-cultured in SOC medium at

37°C for 40 minutes. 50 L of the transformed DH5a cells were plated on a carbenicillin

plate and cultured at 37°C for 12 hours or more. 6 of the produced colonies were selected,

seeded into a carbenicillin-containing LB medium, and suspension-cultured at 37°C at

220 rpm for 12 hours or more. From the plasmid-containing cells, the plasmid was

separated using a plasmid mini kit (Cat.No.27405, QIAGEN). The separated plasmid

was identified by DNA sequencing.

Table 10 below shows the amino acid sequences of TFPI KPI-2 (Kunitz domain

2) for each animal type.

Table 10 Types AA Sequences SEQ ID NOS: Human KPDFCFLEEDPGICRGYITR 30 YFYNNQTKQCERFKYGGC

LGNMNNFETLEECKNICE DG Rabbit KPDFCFLEEDPGICRGFMT 31 RYFYNNQSKQCEQFKYGG CLGNSNNFETLEECRNTCE DP Mouse RPDFCFLEEDPGLCRGYM 32 KRYLYNNQTKQCERFVYG GCLGNRNNFETLDECKKI CENP

Table 11 below shows the primers used in the TFPI KPI-2 (Kunitz domain 2)

gene cloning of Example 5.

Table 11 Names DNA Sequences SEQ ID NOS:

HTK2 For CCATGGAAACCCGACTTT 33 TGCTTCCTGGA RTK2 For CCATGGAAACCCGATTTC 34 TGCTTTCTGGAG MTK2 For CCATGGAGACCTGACTTC 35 TGCTTTCTGGAG HTK2 Re GCGGCCGCCTAGCCGTCT 36 TCACAGATGTTCTTG RTK2 Re GCGGCCGCCTAGGGGTCC 37 TCACAGGTGTTG MTK2 Re GCGGCCGCCTAGGGGTTC 38 TCACAGATTTTCTTGCATT

5-2: Production and Purification of Human TFPI KPI-2 (Kunitz domain 2), Rabbit TFPI

KPI-2 and Mouse TFPI KPI-2 Proteins

The clones with identified TFPI gene sequences were transformed into

BL21(DE3) bacteria (chemical competent cell; Cat.No.C25271, NEB). Each of human

TFPI KPI-2 (Kunitz domain 2), rabbit KPI-2 and mouse KPI-2 genes was added to the bacterial cells which were then incubated on ice for about 10 minutes. For heat shock, the cells were incubated at 42°C for 1 minute, and for cell recovery, the cells were suspension-cultured in SOC at 37°C for 40 minutes. 50 L of the transformed bacterial cells were plated on a carbenicillin plate and cultured at 37°C for 12 hours or more. One of the produced colonies was seeded into a carbenicillin-containing LB medium and suspension-cultured at 37°C at 220 rpm for 12 hours or more. On the next day, the cultured bacterial cells were seeded into 500 ml of SB-glucose medium and suspension cultured at 37°C at 220 rpm for 2 hours. When the OD of the bacterial culture medium reached 0.6, 0.1 mM IPTG was added using NanoDrop for induction. Next, the cells were suspension-cultured at 25°C at 180 rpm for 12 hours or more. The bacterial cells were recovered by centrifugation at 6000 rpm for 20 minutes, and freezing and thawing were repeated three times to recover the protein expression in the periplasm region, followed by centrifugation. The supernatant was passed through a 0.22 m filter to remove cell debris, followed by purification. The purification process was performed using Talon metal affinity resin (Cat.No.635501, Clonetech), and the resin was stabilized with phosphate buffer and incubated with the filtered culture medium at 4°C for 12 hours or more. A washing process was performed using 10 mM imidazole, and an elution process was performed using 250 mM imidazole. The purified protein was electrophoresed on NuPAGE 4-12% Bis-Tris gel, and then the isolated protein was visualized by Coomassie blue staining. The eluted protein was filtered through a

Vivaspin (Cat.No.28-9322-18, GE) column, and the buffer was replaced with PBS

(phosphate-buffered saline) buffer.

As a result, as shown in FIG. 7, protein electrophoresis (SDS-PAGE) indicated

that the TFPI KPI-2 (Kunitz domain 2) protein for each animal type was purified in a

good state.

Example 6: Measurement of Quantitative Affinity for Anti-TFPI Antibody for TFPI

Antigen

The quantitative affinity of clone T417, clone T308, clone 308, clone 308-2 or

clone 308-4, which is the purified anti-TFPI antibody, for recombinant human TFPI, was

measured using a Biacore T-200 (GE Healthcare, USA) biosensor. TFPI (Cat.No.TFPI

875H, Creative Biomart, USA) purified from HEK293 cells was immobilized on a CM5

chip (GE Healthcare, EI-q|) to an Rmax of 200 by an amine-carboxyl reaction, and then

the clone T417, clone T308, clone 308, clone 308-2 or clone 308-4 antibody serially

diluted in HBS-EP buffer (10mM HEPES, pH7.4, 150mM NaCl, 3mM EDTA, 0.005%

surfactant P20) was run on the chip at a concentration of 0.078-10 nM at a flow rate 30

[L/min for 120 seconds for association and 600 seconds for dissociation (Table 12). 10

mM of glycine-HC (pH 1.5) was run at a flow rate of 30 L/min for 30 seconds, thereby

inducing the dissociation of the antibody associated with the TFPI. The affinity in terms

of kinetic rate constants (K.n and Kff) and equilibrium dissociation constant (KD) was

evaluated using Biacore T-200 evaluation software.

As a result, as shown in Table 13 below and FIG. 8, it was shown that the

affinities of the prepared clone 308-2 and clone 308-4 antibodies were higher than that of

clone 308.

Table 12 SPR Biacore T200 Chip CM5 Running Buffer HBS-EP pH7.4 Flow rate 30ul/min Association / dissociation time 120sec / 600sec IgG Conc. 0.3125-5nM, 12serial dilution Regeneration 10mM Glycine-HCl pH1.5, 30sec

Table 13

K Kff K,

T417 5.3X1O6 3.5X1O- 5 6.7X10-2 2 T308 4.4X10 6 4.2X1O-5 9.4X10- 4 308 3.5X106 1.7X10- S.OX1O-11

3.0X106 11 308-2 9.9X10-5 3.3X10-

308-4 3.5X106 8.2X10-5 2.4X10-11

Example 7: Measurement of Fxa Activity

Blood coagulation is induced by an intrinsic pathway and an extrinsic pathway,

and the two pathways activate thrombin through a common pathway that activates factor

X, thereby forming fibrin to induce blood coagulation. In addition, TFPI consists of

Kunitz 1 (KI), Kunitz 2 (K2) and Kunitz 3 (K3)domains. It is known that the KI

domain binds to FVIIa and the K2 domain binds to FXa. It is known that blood

coagulation is inhibited by the binding between TFPI and the blood clotting factor. Thus,

in order to determine the effects of anti-TFPI candidate antibodies on the blood

coagulation process, the FXa activity was evaluated. An assay system was composed

only of FXa, TFPI and a candidate antibody so as to minimize the effects of several

factors. When the candidate antibody binds to TFPI, it does not inhibit the function of

FXa, and thus the FXa activity appears. However, when the candidate antibody does not

effectively bind to TFPI, TFPI binds to FXa to thereby inhibit the function of FXa, and

thus the degree of color development decreases. Thus, the residual activity of FXa which

is not inhibited by TFPI is measured by the degree of substrate degradation. The

substrate used herein is the FXa-specific substrate S-2765, and the substrate is degraded

to generate measurable chromophoric pNA at 405 nm. This measurement method is

based on an amidolytic assay.

Each of FXa, TFPI, mAb2021 and S-2765 was diluted with an assay buffer

(20mM HEPES, 150mM NaCl, 1mg/mL BSA, 0.02% NaN3, 5mM CaCl2, pH7.4) with

reference to Table 14 below and dispensed in a 1.5 ml tube.

Table 14

materials) Pre-dilution Working conc.(nM) Others Conc.(nM) FXa 2nM 0.5nM TFPI 40nM 1OnM S-2765 2mM 0.5mM Standard curve 10nM 0.02, 0.1, 0.5, 2.5nM FXa mAb2021 160nM 0.625, 2.5, 10, 40nM Positive Control

50 pL of each of the positive control (mAb2021, anti-TFPI Ab, Novo Nordisk)

and the anti-TFPI candidate antibodies was added to each well to a concentration of 40,

10, 2.5 or 0.625 nM. 50 L of 40 nM TFPI solution was added to each well and allowed

to stand at room temperature for 30 minutes. To obtain a standard curve, 50 pL of FXa

solution was added to each well at varying concentrations, and 50 L of 2 mM FXa

solution was added to each well and incubated at 37°C for 10 minutes. 50 L of 2 mM S

2765 solution was added to each well and incubated at 37°C for 30 minutes. Then, the

absorbance of each well at a wavelength of 405 nm was read by a microplate reader in

endpoint mode.

As a result, as shown in FIG. 9, both clone T308 and clone T417 that are

chimeric antibodies among the anti-TFPI candidate antibodies showed increases in the

absorbance in an antibody concentration-dependent manner, indicating that the TFPI

inhibitory effects of the two antibodies increase in a concentration-dependent manner.

Clone T308 showed the effect of inhibiting TFPI by 91% in the sample treated with 40

nM, and the effect of inhibiting TFPI by 89% in the sample treated with 10 nM, compared to the sample not treated with TFPI, which is the positive control (mAb2021, anti-TFPI Ab). Clone T417 showed the effect of inhibiting TFPI by 89% in the sample treated with 40 nM, and the effect of inhibiting TFPI by 72% in the sample treated with

10 nM, compared to the sample not treated with TFPI, which is the positive control

(mAb2021, anti-TFPI Ab). When the effects were compared at a TFPI concentration of

10 nM, it could be seen that clone T417 has a better TFPI inhibitory activity than clone

T308.

In addition, as shown in FIG. 10, clone 308 was obtained by humanization of

clone T417 determined to have a better effect in the above assay. Clone 308 also showed

an increase in the absorbance in a concentration-dependent manner, indicating that it

could inhibit TFPI. Clone 308 showed a TFPI inhibitory activity of about 85.1% in the

sample treated with 40 nM, and a TFPI inhibitory activity of about 58.2% in the sample

treated with 10 nM, compared to the positive control (mAb2021, anti-TFPI Ab),

indicating that it has an inferior effect to clone T417 that showed a TFPI inhibitory

activity of 78.4% in the sample treated with 10 nM.

In addition, as shown in FIG. 11, back mutation was performed in order to

be seen that both clone 308-2 and clone 308-4 inhibited TFPI in a concentration

dependent manner. Also, in the samples treated with 40 nM and 10 nM, it could be seen

that the TFPI inhibitory activities of clone 308-2 and clone 308-4 increased compared to

that of clone 308. At a concentration of 40 nM, clone 308-2 and clone 308-4 showed

TFPI inhibitory activities of 85% and 82%, respectively, compared to the positive control

(mAb202l, anti-TFPI Ab), but at a concentration of 10 nM, clone 308-2 showed a TFPI

inhibitory activity of 72%, and clone 308-4 showed a TFPI inhibitory activity of 78%,

which is higher than that of clone 308-2. Additionally, it was shown that these antibodies were comparable to the clone T417 chimeric antibody showing a TFPI inhibitory activity of 77%.

Example 8: Measurement of TF/FVIIa/FXa Complex

The most important factors in the extrinsic pathway of blood coagulation include

TF (tissue factor), FVII (factor VII), FX (factor X) and the like. When TF and FVIIa

form a complex by an external signal, FX is activated into FXa. Then, FXa activates

prothrombin into thrombin, which then converts fibrinogen into fibrin which acts on

blood coagulation. However, TFPI (tissue factor pathway inhibitor) inhibits the function

of FXa by binding to FXa, thereby interfering with blood coagulation. In order to

evaluate the effect of anti-TFPI antibodies in the above-described pathway, a

TF/FVIIa/FXa complex assay was performed. In a state in which TFPI was present

together with or independently of anti-TFPI antibodies, the extents of production and

inhibition of FXa by a TF/FVIIa complex were measured based on the extent of color

development of a substrate (S2765) degraded by FXa, thereby evaluating the effect of the

anti-TFPI antibody. In other words, as the TFPI inhibitory effect of the anti-TFPI

antibody increases, the production of FXa increases, and the amount of substrate

degraded increases, resulting in an increase in absorbance.

In 1.5 mL tubes, TF (4500L/B, Sekisui diagnostics), FVIIa (Novo Nordisk, Novo

Seven), and FX (PP008A, Hyphen biomed)were diluted with assay buffer (20 mM

HEPES, 150 mM NaCl, 1 mg/mL BSA, 0.02% NaN3, 5 mM CaC2, pH 7.4) to the

concentrations shown in Table 15 below, thereby preparing a mixture solution.

Table 15 Material TF FVIIa FX Conc. 6ng/mL 800nM 30nM

70 pL of the mixture solution was added to each well of a 96-well plate. To a

blank well, 70 L of assay buffer was added. Each well was incubated at 37°C for 15 minutes, and then 30 L of TFPI was added to each well to a concentration of 50 nM.

However, 30 L of assay buffer was added to each of the blank well and a positive

control well (a sample not treated with the anti-TFPI antibody and TFPI). 30 L of the

anti-TFPI antibody was added to each well to concentrations of 12.5, 25, 50 and 100 nM.

To each of the blank well, the positive control well (a sample not treated with the anti

TFPI antibody and TFPI) and the negative control well (a sample not treated with the

anti-TFPI antibody), 30 L of assay buffer was added, followed by incubation at 37°C for

15 minutes. 20 L of EDTA (E7889, Sigma-Aldrich) was added to each well to a

concentration of 50 mM. Next, 50 L of S2765 was added to each well to a

concentration of 200 [M, followed by incubation at 37°C for 10 minutes. Next, the

absorbance of each well at 405 nm was measured using a microplate reader.

As a result, as shown in FIG. 12, the effects of clone T308 and clone T417 that

are chimeric antibodies among the anti-TFPI candidate antibodies were confirmed. It

was shown that the two antibodies all showed an increase in the absorbance in an

antibody concentration-dependent manner, indicating that the TFPI inhibitory effects of

the two antibodies increase in a concentration-dependent manner. Clone T308 showed

the effect of inhibiting TFPI by 100% in the sample treated with 100 nM, and the effect

of inhibiting TFPI by about 87% in the sample treated with 50 nM, compared to the

positive control (the sample not treated with the anti-TFPI antibody and TFPI). Clone

T417 showed the effect of inhibiting TFPI by 100% in the samples treated with 100 nM

and 50 nM, compared to the positive control (the sample not treated with the anti-TFPI

antibody and TFPI). Thus, it could be seen that the TFPI inhibitory activity of clone

T417 is higher than that of clone T308.

In addition, as shown in FIG. 13, clone 308 was obtained by humanization of the

clone T417 antibody having a better effect than clone T308. Clone 308 also showed an increase in the absorbance in a concentration-dependent manner, indicating that it inhibited TFPI. Clone 308 showed TFPI inhibitory activities of about 94.3% in the sample treated with 100 nM and about 54.2% in the sample treated with 50 nM, compared to the positive control (the sample not treated with the anti-TFPI antibody and

TFPI), indicating that the effect of clone 308 is inferior to that of clone T417 showing a

TFPI inhibitory activity of 100%.

Furthermore, as shown in FIG. 14, back mutation was performed in order to

increase the effect of the humanized clone 308 antibody, and clone 308-2 and clone 308

4 were obtained. It could be seen that both clone 308-2 and clone 308-4 inhibited TFPI

in a concentration-dependent manner. In addition, in the samples treated with 50 nM, it

could be seen that the TFPI inhibitory activities of clone 308-2 and clone 308-4 increased

compared to that of clone 308. At concentrations of 100 nM and 50 nM, clone 308-2 and

clone 308-4 all showed a TFPI inhibitory activity of 100% compared to the positive

control (the sample not treated with the anti-TFPI antibody and TFPI). At a

clone 308-4 showed a TFPI inhibitory activity of 68.4%, which is higher than that of

clone 308-2. However, it could be seen that the TFPI inhibitory activities of the back

mutated antibodies were lower than that the clone T417 chimeric antibody.

Example 9: Measurement of Thrombin Generation

The blood coagulation mechanism is divided into an intrinsic pathway and an

extrinsic pathway. It is known that the function of TF (tissue factor) in the extrinsic

pathway is the activity feedback function in the blood coagulation mechanism and is the

explosive production of thrombin that is produced very fast. The most important factors

in this blood coagulation mechanism include TF (tissue factor), FVII (factor VII), FX

(factor X) and the like. When TF and FVIIa form a complex by an external signal, FX is activated into FXa. Then, FXa activates prothrombin into thrombin, which then cleaves fibrinogen into fibrin which acts on blood coagulation. However, TFPI (tissue factor pathway inhibitor) acts to inhibit the function of FXa by binding to FXa, thereby interfering with blood coagulation. A thrombin generation assay comprises: treating plasma with a test sample to be evaluated; and then inspecting the amount of thrombin produced in the plasma, based on the amount of a fluorescent product produced when the produced thrombin converts a fluorogenic substrate into the fluorescent product in the presence of PPP-reagent low; and calibrating the inspected amount of thrombin with the known amount of thrombin calibrator, thereby measuring the actual generation of thrombin.

20 L of PPP-reagent low solution was added to the sample loading well of a

prewarmed 96-well plate (round bottom immulon 2HB 96 well plate), and 20 L of

calibrator solution was added to the calibrator well of the plate. An anti-TFPI candidate

antibody was diluted in a pre-dissolved sample dilution (FVIII-deficient plasma) at a

concentration of 0.3125, 0.625, 1.25 or 2.5 nM, and then incubated at room temperature

for 10 minutes so that it could bind to TFPI.

80 L of each of the sample dilution was added to each of the calibrator and

blank wells, and 80 L of the diluted antibody solution was added to each of the

remaining wells. A start button at the bottom of the software screen was pressed to

execute washing. Washing was performed in a state in which an inlet tube was placed in

distilled water in a water bath at 37°C and in which an outlet tube was placed in an empty

container. After completion of the washing, the next button was pressed to perform an

empty process. The inlet tube was placed in a FluCa solution warmed to 37°C and was

primed to fill the tube with the solution. The outlet tube was mounted in an M hole in a

dispenser, and then the next button was pressed to automatically dispense 20 L of FluCa solution into each well, after which a shaking process was performed and analysis was initiated.

As a result, as shown in FIG. 15, a thrombin generation assay was performed

using the clone T417 chimeric antibody and humanized clone 308 antibody selected

through the above-described Fxa activity assay and TF/FVIIa/FXa complex assay. At 2.5

nM, clone T417 showed an increase in thrombin peak of 208% compared with the blank

treated with only the sample dilution, and clone 308 showed an increase in thrombin peak

of 162% compared to the blank. In the case of ETP indicating the total generation of

thrombin, in the samples treated with 2.5 nM, clone T417 showed an increase in ETP of

131%, and clone 308 showed an increase in ETP of 122%, compared to the negative

control (having no antibody). When the two antibodies were compared, it was shown

that clone T417 has a better effect than the clone 308 antibody.

In addition, as shown in FIG. 16, for the clone 308-2 and clone 308-4 antibodies

selected through the FXa activity assay and the TF/FVIIa/FXa complex assay after

performing back mutation in order to increase the effect of the humanized clone 308

antibody, a thrombin generation assay was performed. It was shown that both clone 308

2 and clone 308-4 showed an increase in thrombin generation in a concentration

dependent manner. When the samples treated with 2.5 nM were compared, it could be

seen that clone 308-2 and clone 308-4 showed increases in thrombin peak and total

thrombin generation compared to the clone 308 antibody. In the samples treated with 2.5

nM, clone 308-2 and clone 308-4 showed increases in thrombin peak of 183% and 191%,

respectively, compared to the negative control (having no antibody), and the ETP value

was 126% in both clone 308-2 and clone 308-4, suggesting that clone 308-2 and clone

308-4 have an increased ability to generate thrombin. In addition, the ability of the two antibodies to generate thrombin was superior to that of the clone 308 antibody and was comparable to that of the clone T417 chimeric antibody.

Example 10: Prediction of Binding between Anti-TFPI Antibody 308-4 Clone and

Kunitz Domain-2

As an antibody against TFPI (tissue factor pathway inhibitor) that inhibits the

activity of factor X, an antibody for treating or preventing hemophilia, which can prevent

the inhibition of blood coagulation, was constructed.

Blood coagulation is induced by an intrinsic pathway and an extrinsic pathway,

Kunitz 1 (K1), Kunitz 2 (K2) and Kunitz 3 (K3) domains. It is known that the K1

domain binds to FVIIa and the K2 domain binds to FXa.

As described in Korean Patent Application No. 10-2015-0026555, entitled

"Novel Anti-TFPI Antibody and Composition Comprising the Same", 308-4 clone that is

an anti-TFPI antibody was prepared. It could be seen that the 308-4 clone has a KD of

2.64x10- 11M or lower, preferably 2.52x10-" M or lower, more preferably 2.4x10-" M or

lower.

In the present invention, it was attempted to prepare an antibody having a higher

affinity for TFPI by affinity maturation of the 308-4 clone.

In order to predict the binding between the anti-TFPI antibody 308-4 clone and

the Kunitz domain-2, homology search was performed in the Igblast

(http://blast.ncbi.nlm.nih.gov/Blast.cgi) using the amino acid sequence of the 308-4 clone.

As a result, it was found that the 3QOS (PDB number) structure is similar. Based on

3QOS, the structure of the 308-4 clone was designed using homology modeling that is

the bioluminate module (Schrodinger, Germany). The designed structure was subjected to docking simulation with Kunitz domain-2 using the protein-protein binding prediction program PIPER to obtain binding prediction data. To select paratopes from the obtained binding structure, the interaction between the 308-4 clone and the Kunitz domain-2 was analyzed, and the amino acids of the 308-4 clone, which produce a non-covalent bond, were selected (Table 16). The selected paratopes were subjected to affinity maturation using the bioluminate module to calculate the binding energy value of each paratope and to predict the binding energy value that would be changed by substitution with other amino acids. Thus, amino acids having stable binding energy values were selected and reflected in the design of primers (Table 17).

Table 16 below shows the selected amino acids of the anti-TFPI antibody 308-4

clone, which were determined to produce a non-covalent bond in the analysis of the

interaction between the 308-4 clone and the Kunitz domain-2.

Table 17 below shows the selected amino acids of Table 16, which were

determined to have the stable binding energy values of paratopes by affinity maturation.

Table 16 Variable regions Selected amino acids (based on kabat)

Heavy chain S31, T52a, Y56, E64, N98

Light chain S31a, T92, H93

Table 17 Variable regions Selected amino acids

VHS31 H, K, R, T, Y, I, L

VHT52a F, Y, L, H, K, R, I

VHY56 H, R, K

VHE64 Q, D, H

VHN98 F, H, K, Q, R, Y

VLS31a I, L, N, Q, R, F, K, T, V

VLT92 F, Y, I, N

VLH93 Y, L, I, Q, N, K

Example 11: Preparation of Novel Antibody by Affinity Maturation of 308-4 Clone

Using Yeast Display scFv Library

11-1: Construction of Yeast Display scFv Library

In order to introduce a mutation into a yeast library, three heavy-chain variable

region fragments and two light-chain variable region fragments were subjected to

polymerase chain reaction (PCR). Specifically, for the PCR of heavy-chain variable

region fragment 1, the heavy-chain variable region gene sequence of the anti-TFPI 308-4

clone was used as a template together with a forward primer (Table 18; SEQ ID NO: 40)

and a reverse primer (Table 18; SEQ ID NOs: 41 to 48); for the PCR of heavy-chain

variable region fragment 2, the heavy-chain variable region gene sequence of the anti

TFPI 308-4 clone was used as a template together with a forward primer (Table 18; SEQ

ID NO: 49) and a reverse primer (Table 18; SEQ ID NOs: 50 to 61); and for the PCR of

heavy-chain variable region fragment 3, the heavy-chain variable region gene sequence

of the anti-TFPI 308-4 clone was used as a template together with a forward primer

(Table 18; SEQ ID NO: 62) and a reverse primer (Table 18; SEQ ID NOs: 63 to 69). The

PCR of each of the fragments was performed using AccuPower Pfu PCR PreMix

(CAT.No.K-2015, Bioneer). The PCR was performed under the following conditions: 2

min at 95°C; and then 30 cycles, each consisting of 30 see at 95°C, 30 see at 55°C and 60

see at 72°C; and followed by 10 min at 72°C. The amplified genes were electrophoresed on 1% agarose gel to confirm the DNA band having the expected size, and were isolated using a gel extraction kit (QAquick Gel Extraction Kit, CAT.No.28706, QIAGEN). For the PCR of light-chain variable region fragment 1, the light-chain variable region gene sequence of the anti-TFPI 308-4 clone was used as a template together with a forward primer (Table 18; SEQ ID NO: 72) and a reverse primer (Table 18; SEQ ID NOs: 73 to

82); and for the PCR of light-chain variable region fragment 2, the light-chain variable

region gene sequence of the anti-TFPI 308-4 clone was used as a template together with a

forward primer (Table 18; SEQ ID NO: 83) and a reverse primer (Table 18; SEQ ID

NOs: 84 to 87). The PCR of each of the fragments was performed using AccuPower Pfu

PCR PreMix (Bioneer) under the following conditions: 2 min at 95°C; and then 30 cycles,

each consisting of 30 see at 95°C, 30 see at 55°C and 60 see at 72°C; and then 10 min at

72°C. The amplified genes were electrophoresed on 1% agarose gel to confirm the DNA

band having the expected size, and were isolated using a gel extraction kit (QAquick Gel

Extraction Kit, QIAGEN).

The obtained heavy-chain variable region fragment genes were adjusted to a

molar ratio of 1:1:1 and used as a template together with a forward primer (Table 18;

SEQ ID NO: 70) and a reverse primer (Table 18; SEQ ID NO: 71) in PCR. The PCR of

the fragment genes was performed using Takara primer star PCR premix

(CAT.NO.R40B, Takara) under the following conditions: 2 min at 95°C; and then 20

cycles, each consisting of 10 see at 95°C, 20 see at 55°C and 30 see at 72°C; and then 5

min at 72°C. The amplified gene was electrophoresed on 1% agarose gel to confirm the

DNA band having the expected size, and was isolated using a gel extraction kit

(QlAquick Gel Extraction Kit, QIAGEN), thereby obtaining a heavy-chain variable

region gene.

The obtained light-chain variable region fragment genes were adjusted to a molar

ratio of 1:1 and used as a template together with a forward primer (Table 18; SEQ ID

NO: 91) and a reverse primer (Table 18; SEQ ID NO: 92) in PCR. The PCR of the

fragment genes was performed using Takara primer star PCR premix (CAT.No.R040B,

Takara) under the following conditions: 2 min at 95°C; and then 20 cycles, each

consisting of 10 see at 95°C, 30 see at 55°C and 40 see at 72°C; and then 5 min at 72°C.

The amplified gene was electrophoresed on 1% agarose gel to confirm the DNA band

having the expected size, and was isolated using a gel extraction kit (QAquick Gel

Extraction Kit, QIAGEN), thereby obtaining a light-chain variable region gene.

The obtained heavy-chain and light-chain variable region genes were adjusted to

a molar ratio of 1:1 and used as a template together with a forward primer (Table 18;

SEQ ID NO: 93) and a reverse primer (Table 18; SEQ ID NO: 94) in PCR. The PCR of

the genes was performed using Takara primer star PCR premix (Takara) under the

following conditions: 2 min at 95°C; and then 20 cycles, each consisting of 10 see at

95°C, 20 see at 55°C and 30 see at 72°C; and then 5 min at 72°C. The amplified gene

was electrophoresed on 1% agarose gel to confirm the DNA band having the expected

size, and was isolated using a gel extraction kit (QAquick Gel Extraction Kit, QIAGEN),

thereby constructing a 308-4 affinity maturation scFv library gene. 200 ng of the

constructed library gene was mixed with 1 pg of the pCTCON gene treated with the

restriction enzymes NheI (CAT.No.R0131L, NEB) and BamHI(CAT.No.R0136L, NEB),

and the mixture was transformed into yeast (EBY100 electro-competent cell). The

transformed yeast was suspended in 100 mL of YPD medium and shake-cultured at 30°C

at 200 rpm for 1 hour. The cultured yeast was inoculated into 1 L of SD medium and

cultured at 30°C at 200 rpm for 12 hours or more, after which it was centrifuged to

remove the supernatant, and resuspended in yeast storage buffer and stored at -70°C. To determine the size of the library, 100 p2 of the culture medium was collected at 1 hour after transformation, plated on SD plate by a serial dilution method, incubated at 30°C for

12 hours or more, and then subjected to colony counting.

Table 18 below shows the primers used in the construction of the yeast display

scFv library.

Table 18 Names Nucleic acid sequences SEQ ID

NOS:

VH FRI Fo GAA GTC CAG CTG GTG GAG TCT GGA GGT 40

VH FRI ReS CGG GGC CTG ACG AAC CCA GTT CAT GGC ATA GCT 41

GCT GAA GGT GAA GCC GCT CGC TGC

VH FRI ReH CGG GGC CTG ACG AAC CCA GTT CAT GGC ATA ATG 42

GCT GAA GGT GAA GCC GCT CGC TGC

VH FRI ReK CGG GGC CTG ACG AAC CCA GTT CAT GGC ATA TTT 43

GCT GAA GGT GAA GCC GCT CGC TGC

VH FRI ReR CGG GGC CTG ACG AAC CCA GTT CAT GGC ATA TCT 44

GCT GAA GGT GAA GCC GCT CGC TGC

VH FRI ReT CGG GGC CTG ACG AAC CCA GTT CAT GGC ATA AGT 45

GCT GAA GGT GAA GCC GCT CGC TGC

VH FRI ReY CGG GGC CTG ACG AAC CCA GTT CAT GGC ATA ATA 46

GCT GAA GGT GAA GCC GCT CGC TGC

VH FRI ReI CGG GGC CTG ACG AAC CCA GTT CAT GGC ATA AAT 47

GCT GAA GGT GAA GCC GCT CGC TGC

VH FRI ReL CGG GGC CTG ACG AAC CCA GTT CAT GGC ATA AAG 48

GCT GAA GGT GAA GCC GCT CGC TGC

VH FR2 Fo TAT GCC ATG AAC TGG GTT CGT CAG GCC 49

VH FR2 ReT- GTT ATC GCG GGA AAT GGT GAA GCG CCC X3TX2 AAC 50

YH-EQDH GCT ATC GGC GTA GTA GGT GTX1 TGA CCC ACC GGT

TGT GAT GGT GCT GAC CCA TTC CAA GCC

VH FR2 ReT- GTT ATC GCG GGA AAT GGT GAA GCG CCC X3TX2 AAC 51

RK-EQDH GCT ATC GGC GTA GTA GGT TYT TGA CCC ACC GGT TGT GAT GGT GCT GAC CCA TTC CAA GCC

VH FR2 GTT ATC GCG GGA AAT GGT GAA GCG CCC X3TX2 AAC 52

ReFYLH-YH- GCT ATC GGC GTA GTA GGT GTX1 TGA CCC ACC TWR

EQDH TGT GAT GGT GCT GAC CCA TTC CAA GCC

VH FR2 GTT ATC GCG GGA AAT GGT GAA GCG CCC X3TX2 AAC 53

ReFYLH-RK- GCT ATC GGC GTA GTA GGT TYT TGA CCC ACC TWR

EQDH TGT GAT GGT GCT GAC CCA TTC CAA GCC

VH FR2 GTT ATC GCG GGA AAT GGT GAA GCG CCC X3TX2 AAC 54

ReKRI-YH- GCT ATC GGC GTA GTA GGT GTX1 TGA CCC ACC THT

EQDH TGT GAT GGT GCT GAC CCA TTC CAA GCC

VH FR2 GTT ATC GCG GGA AAT GGT GAA GCG CCC X3TX2 AAC 55

ReKRI-RK- GCT ATC GGC GTA GTA GGT TYT TGA CCC ACC THT

EQDH TGT GAT GGT GCT GAC CCA TTC CAA GCC

VH FR2 ReT- GTT ATC GCG GGA AAT GGT GAA GCG CCC NTS AAC 56

YH-EQDH_#2 GCT ATC GGC GTA GTA GGT ATR TGA CCC ACC GGT

TGT GAT GGT GCT GAC CCA TTC CAA GCC

VH FR2 ReT- GTT ATC GCG GGA AAT GGT GAA GCG CCC NTS AAC 57

RK-EQDH_#2 GCT ATC GGC GTA GTA GGT TYT TGA CCC ACC GGT

TGT GAT GGT GCT GAC CCA TTC CAA GCC

VH FR2 GTT ATC GCG GGA AAT GGT GAA GCG CCC NTS AAC 58

ReFYLH-YH- GCT ATC GGC GTA GTA GGT ATR TGA CCC ACC TWR

EQDH_#2 TGT GAT GGT GCT GAC CCA TTC CAA GCC

VH FR2 GTT ATC GCG GGA AAT GGT GAA GCG CCC NTS AAC 59

ReFYLH-RK- GCT ATC GGC GTA GTA GGT TYT TGA CCC ACC TWR

EQDH_#2 TGT GAT GGT GCT GAC CCA TTC CAA GCC

VH FR2 GTT ATC GCG GGA AAT GGT GAA GCG CCC NTS AAC 60

ReKRI-YH- GCT ATC GGC GTA GTA GGT ATR TGA CCC ACC THT

EQDH_#2 TGT GAT GGT GCT GAC CCA TTC CAA GCC

VH FR2 GTT ATC GCG GGA AAT GGT GAA GCG CCC NTS AAC 61

Re_KRI-RK- GCT ATC GGC GTA GTA GGT TYT TGA CCC ACC THT

EQDH_#2 TGT GAT GGT GCT GAC CCA TTC CAA GCC

VH FR3 Fo GGG CGC TTC ACC ATT TCC CGC GAT AAC 62

VH FR3 ReN GCC CTG GCC CCA ATA ATC CAT CAG AAA ATT GCC 63

ATC CTG GCG CGC GCA ATA ATA TAC CGC

VH FR3 ReF GCC CTG GCC CCA ATA ATC CAT CAG AAA AAA GCC 64

ATC CTG GCG CGC GCA ATA ATA TAC CGC

VH FR3 ReH GCC CTG GCC CCA ATA ATC CAT CAG AAA ATG GCC 65

ATC CTG GCG CGC GCA ATA ATA TAC CGC

VH FR3 ReK GCC CTG GCC CCA ATA ATC CAT CAG AAA TTT GCC 66

ATC CTG GCG CGC GCA ATA ATA TAC CGC

VH FR3 ReQ GCC CTG GCC CCA ATA ATC CAT CAG AAA TTG GCC 67

ATC CTG GCG CGC GCA ATA ATA TAC CGC

VH FR3 ReR GCC CTG GCC CCA ATA ATC CAT CAG AAA TCT GCC 68

ATC CTG GCG CGC GCA ATA ATA TAC CGC

VH FR3 ReY GCC CTG GCC CCA ATA ATC CAT CAG AAA ATA GCC 69

ATC CTG GCG CGC GCA ATA ATA TAC CGC

VH Final Fo GGT TCT GGT GGT GGT GGT TCT GCT AGC GAC GTG 70

GTGATGACACAGACGCCGCTG

VH Final Re GGA GCT CAC AGT CAC CAG CGT GCC CTG GCC CCA 71

ATA ATC CAT CAG AAA

VL FRI Fo GAC GTG GTG ATG ACA CAG ACG CCG CTG 72

VL FRI ReS GAG CCA ATT CAG ATA CGT CTT GCC GTC GGA GTC 73

CAGCAGCGACTGGCTTGATTTGCA

VL FR IReI GAG CCA ATT CAG ATA CGT CTT GCC GTC AAT GTC 74

CAGCAGCGACTGGCTTGATTTGCA

VL FR IReL GAG CCA ATT CAG ATA CGT CTT GCC GTC AAG GTC 75

CAGCAGCGACTGGCTTGATTTGCA

VL FRI ReN GAG CCA ATT CAG ATA CGT CTT GCC GTC AGC GTC 76

CAGCAGCGACTGGCTTGATTTGCA

VL FRI ReQ GAG CCA ATT CAG ATA CGT CTT GCC GTC TTG GTC 77

CAGCAGCGACTGGCTTGATTTGCA

VL FRI ReR GAG CCA ATT CAG ATA CGT CTT GCC GTC TCT GTC 78

CAGCAGCGACTGGCTTGATTTGCA

VL FRI ReF GAG CCA ATT CAG ATA CGT CTT GCC GTC AAA GTC 79

CAGCAGCGACTGGCTTGATTTGCA

VL FRI ReK GAG CCA ATT CAG ATA CGT CTT GCC GTC TTT GTC 80

CAGCAGCGACTGGCTTGATTTGCA

VL FRI ReT GAG CCA ATT CAG ATA CGT CTT GCC GTC AGT GTC 81

CAGCAGCGACTGGCTTGATTTGCA

VL FRI ReV GAG CCA ATT CAG ATA CGT CTT GCC GTC AAC GTC 82

CAGCAGCGACTGGCTTGATTTGCA

VL FR2 Fo GAC GGC AAG ACG TAT CTG AAT TGG CTC CAG 83

VL FR2 ReT- GCG TTT AAT TTC AAC CTT AGT GCC TTG GCC GAA 84

YH CGT AAA CGG AAA GTR GGT GCC CTG CCA GCA ATA GTA GAC GCC

VL FR2 ReT- GCG TTT AAT TTC AAC CTT AGT GCC TTG GCC GAA 85

LIHQNK CGT AAA CGG AAA WWK GGT GCC CTG CCA GCA ATA GTA GAC GCC

VL FR2 GCG TTT AAT TTC AAC CTT AGT GCC TTG GCC GAA 86

ReFYIN-YH CGT AAA CGG AAA GTR AWW GCC CTG CCA GCA ATA

GTA GAC GCC

VL FR2 GCG TTT AAT TTC AAC CTT AGT GCC TTG GCC GAA 87

ReFYIN- CGT AAA CGG AAA WWK AWW GCC CTG CCA GCA ATA

LIHQNK GTA GAC GCC

VL Final Re GCG TTT AAT TTC AAC CTT AGT GCC TTG GCC GAA 88

CGT AAA

VL Final Fo SfiI Cgtggcccaggcggcc GAC GTG GTG ATG ACA CAG ACG CCG 89

CTG

VL Final Fo NruI Cta TCG CGA TTG CAG TGG CAC TGG CTG GTT TCG 90

VL Overlapping GGC ACG CTG GTG ACT GTG AGC TCC Gga ggc ggc gga agt 91

Fo ggc gga gga ggc age ggc gga ggc ggg agt GAC GTG GTG ATG

ACACAGACGCCGCTG

VL Final Re GTC CTC TTC AGA AAT AAG CTT TTG TTC GGA TCC 92

GCG TTT AAT TTC AAC CTT AGT GCC TTG GCC GAA CGT AAA

VH Homologous GCT CTG CAG GCT AGT GGT GGT GGT GGT TCT GGT 93

recombination GGT GGT GGT TCT GGT GGT GGT GGT TCT get age

VL Homologous TTG TTA TCA GAT CTC GAG CTA TTA CAA GTC CTC TTC 94

recombination AGA AAT AAG CTT TTG TTC GGA TCC

11-2: Antibody Selection

The library yeast cells constructed in Example 11-1 were inoculated into SD

medium and cultured at 30°C at 200 rpm for 12 hours or more, and then the medium was

replaced with SG medium, and the cells were cultured at 25°C at 200 rpm for 12 hours to

express the antibody on the yeast surface. Next, the yeast cells collected by

centrifugation were washed with PBSM (3% BSA containing PBS) buffer, resuspended

in 1 mL of PBSM buffer and incubated with a biotin-conjugated recombinant human

TFPI protein at room temperature for 1 hour. The yeast cells incubated with the

recombinant human TFPI protein were washed with PBSM, and then incubated with

streptavidin microbeads (CAT.NO.130-048-101, Miltenyi biotech) on ice for 15 minutes.

Next, the cells were washed once with PBSM buffer, resuspended in PBSM buffer, and

then passed through an MACS column (CAT.NO.130-042-901, Milternyi biotech) to

separate TFPI protein-conjugated yeast cells. The separated yeast cells were inoculated

into SD medium and cultured for 48 hours or more, and the above procedure was

repeated twice, thereby selecting the antibody.

11-3: Preparation of Individual Clones by FACS

The finally amplified single colonies were collected from the yeast display

library, and then cultured in SD medium at 30°C at 200 rpm for 12 hours. Then, the

medium was replaced with SG medium, and the cells were cultured at 25°C at 200 rpm

for 12 hours or more, thereby expressing the antibody on the yeast surface. Next, the

yeast cells recovered by centrifugation were washed with PBSF (1% BSA containing

PBS) buffer, resuspended in 50 p2 of PBSF buffer, and then incubated with a biotin

conjugated recombinant human TFPI protein and anti-c-myc mouse antibody

(CAT.No.M4439, Sigma) at room temperature for 30 minutes. The incubated yeast cells

were washed with PBSF, resuspended in 50 p2 of PBSF buffer, and then incubated with

FITC-conjugated anti-mouse antibody (CAT.No.F0257, Sigma) and PE-conjugated

streptavidin on ice under a light-shielded condition for 15 minutes. Next, the cells were

washed with PBSF buffer, resuspended in 500 p2 of PBSF buffer, and then clones

showing high values in the FITC and PE wavelength ranges were selected by FACS,

thereby obtaining individual clones.

As a result, as shown in Table 19 below, clones that bind specifically to human

TFPI could be selected, and the amino acid sequences thereof were analyzed. Among the

antibodies described in Korean Patent Application No. 10-2015-0026555, the antibody

used in the present invention was described as '2015-26555_(SEQ ID NO of the previous

application)'.

Table 20 below shows the CDR amino acid sequences of the clone antibodies of

Table 19, identified based on the Kabat numbering system.

Table 19 Variable SEQ ID Clones Amino acid sequences regions NOS:

1001 Heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 95

RQAPGKGLEWVSTITTGGSYTYYADSVDGRFTISRDN AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDIDGKTYL 96

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

1015 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 97

Chain RQAPGKGLEWVSTITTGGSHTYYADSVEGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDIDGKTYL 96

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

1021 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 98

Chain RQAPGKGLEWVSTITTGGSYTYYADSVDGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGHFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDIDGKTYL 96

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

1023 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 99

Chain RQAPGKGLEWVGTITTGGSYTYYADSVDGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDIDGKTYL 96

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

1024 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWV 100

Chain RQAPGKGLEWVSTITTGGSYTYYADSVDGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDLDGKTYL 101

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

1104 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 102

Chain RQAPGKGLEWVGTITTGGSHTYYADSVQGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDVDGKTYL 103

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

1123 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 104

Chain RQAPGKGLEWVSTITTGGSHTYYADSVEGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGHFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDIDGKTYL 96

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

1202 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 105

Chain RQAPGKGLEWVGTITTGGSYTYYADSVDGRFTISRDN

AKNSLYLKMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDIDGKTYL 96

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

1208 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 104

Chain RQAPGKGLEWVSTITTGGSHTYYADSVEGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGHFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDVDGKTYL 106

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTYLPFTFGQGTKVEIKR

1214 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 2015

Chain RQAPGKGLEWVSTITTGGSYTYYADSVEGRFTISRDN 26555_(25)

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDVDGKTYL 103

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

1216 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSHYAMNW 107

Chain VRQAPGKGLEWVSTITTGGSYTYYADSVEGRFTISRD

NAKNSLYLQMNSLRAEDTAVYYCARQDGHFLMDYW GQGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDIDGKTYL 108

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHLPFTFGQGTKVEIKR

1223 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 109

Chain RQAPGKGLEWVSTITTGGSYTYYADSVEGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGHFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDIDGKTYL 96

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

1224 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWV 100

Chain RQAPGKGLEWVSTITTGGSYTYYADSVDGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDIDGKTYL 96

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

1232 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 98

Chain RQAPGKGLEWVSTITTGGSYTYYADSVDGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGHFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDVDGKTYL 103

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

1234 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 110

Chain RQAPGKGLEWVSTITTGGSYTYYADSVQGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDIDGKTYL 111

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLEISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

1238 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 109

Chain RQAPGKGLEWVSTITTGGSYTYYADSVEGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGHFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDVDGKTYL 103

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

1243 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 112

Chain RQAPGKGLEWVSTITTGGSHTYYADSVHGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGHFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDIDGKTYL 96

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

1248 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 113

Chain RQAPGKGLEWVSTITTGGSHTYYADSVDGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGHFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDIDGKTYL 96

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

3007 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFTSYAMNWV 114

Chain RQAPGKGLEWVSTITLGGSYTYYADSVQGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDLDGKTYL 101

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

3016 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 115

Chain RQAPGKGLEWVSTITTGGSHTYYADSVEGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

LightChain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDSDGKTYL 116

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHLPFTFGQGTKVEIKR

3024 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWV 117

Chain RQAPGKGLEWVSTITTGGSYTYYADSVQGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDSDGKTYL 116

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHLPFTFGQGTKVEIKR

3115 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 117

Chain RQAPGKGLEWVGTITTGGSHTYYADSVDGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDVDGKTYL 103

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

3120 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 118

Chain RQAPGKGLEWVSTITTGGSYTYYADSVEGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGYFLMDYWG QGTLVTVSS

LightChain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDIDGKTYL 96

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

3131 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWV 119

Chain RQAPGKGLEWVSTITTGGSYTYYADSVQGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGQFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDIDGKTYL 96

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

3203 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWV 120

Chain RQAPGKGLEWVSTITTGGSYTYYADSVEGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDLDGKTYL 101

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

3241 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 2015

Chain RQAPGKGLEWVSTITTGGSYTYYADSVEGRFTISRDN 26555_(25)

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

LightChain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDIDGKTYL 96

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

4011 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFYSYAMNW 121

Chain VRQAPGKGLEWVSTITTGGSHTYYADSVEGRFTISRD

NAKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYW GQGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDVDGKTYL 122

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHLPFTFGQGTKVEIKR

4017 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 123

Chain RQAPGKGLEWVGTITTGGSYTYYADSVQGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGYFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDIDGKTYL 96

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

4034 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 124

Chain RQAPGKGLEWVSTITTGGSHTYYADSVEGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGYFLMDYWG QGTLVTVSS

LightChain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDIDGKTYL 96

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

4041 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWV 125

Chain RQAPGKGLEWVSTITTGGSHTYYADSVEGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGYFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDVDGKTYL 103

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

4141 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 126

Chain RQAPGKGLEWVGTITTGGSHTYYADSVEGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGYFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDVDGKTYL 103

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

4146 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 127

Chain RQAPGKGLEWVSTITTGGSYTYYADSVEGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGYFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDIDGKTYL 96

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

4206 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 128

Chain RQAPGKGLEWVSTITTGGSHTYYADSVEGRFTISRDN

AKNSLYLQMDSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDVDGKTYL 122

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHLPFTFGQGTKVEIKR

4208 Heavy EVQLVESGGGLVKSGGSLRLSCAASGFTFSSYAMSWV 129

Chain RQAPGKGLEWVGTITTGGSYTYYADSVQGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDTDGKTYL 130

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

4278 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSKYAMNWF 131

Chain RQAPGKGLEWVSTITLGGSYTYYADSVDGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQYLDGNFLMDY WGQGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDIDGKTYL 96

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

4287 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSKYAMNWF 132

Chain RQAPGKGLEWVSTITTGGSYTYYADSVEGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQHPYGNFLMDY WGQGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDIDGKTYL 96

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 99

Chain RQAPGKGLEWVGTITTGGSYTYYADSVDGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDSDGKTYL 2015

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF 26555_(22)

TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

2 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 2015

Chain RQAPGKGLEWVSTITTGGSYTYYADSVEGRFTISRDN 26555_(25)

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDIDGKTYLN 96

WLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFT LKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

3 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 117

Chain RQAPGKGLEWVGTITTGGSHTYYADSVDGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDVDGKTYL 103

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

4 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 133

Chain RQAPGKGLEWVGTITTGGSHTYYADSVEGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDVDGKTYL 103

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 95

Chain RQAPGKGLEWVSTITTGGSYTYYADSVDGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDSDGKTYL 2015

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF 26555_(22)

TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

6 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 134

Chain RQAPGKGLEWVGTITTGGSYTYYADSVEGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDSDGKTYL 2015

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF 26555_(22)

TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

7 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 99

Chain RQAPGKGLEWVGTITTGGSYTYYADSVDGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDVDGKTYL 103

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

8 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 2015

Chain RQAPGKGLEWVSTITTGGSYTYYADSVEGRFTISRDN 26555_(25)

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDVDGKTYL 103

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

9 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 135

Chain RQAPGKGLEWVGTITTGGSYTYYADSVQGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDVDGKTYL 103

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 110

Chain RQAPGKGLEWVSTITTGGSYTYYADSVQGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDVDGKTYL 103

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

11 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 136

Chain RQAPGKGLEWVGTITTGGSYTYYADSVEGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDIDGKTYLN 96

WLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFT LKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

12 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 137

Chain RQAPGKGLEWVGTITTGGSHTYYADSVEGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGHFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDIDGKTYLN 96

WLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFT LKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

13 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 138

Chain RQAPGKGLEWVGTITTGGSHTYYADSVEGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDIDGKTYLN 96

WLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFT LKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

14 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSKYAMNWF 131

Chain RQAPGKGLEWVSTITLGGSYTYYADSVDGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQYLDGNFLMDY WGQGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDSDGKTYL 139

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTYFPFTFGQGTKVEIKR

Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSKYAMNWF 132

Chain RQAPGKGLEWVSTITTGGSYTYYADSVEGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQHPYGNFLMDY WGQGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDSDGKTYL 140

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGFYFPFTFGQGTKVEIKR

16 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSHYAMTWV 141

Chain RQAPGKGLEWVSTITTGGSHTYYADSVEGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDIDGKTYLN 96

WLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFT LKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

17 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMTWV 142

Chain RQAPGKGLEWVSTITTGGSHTYYADSVQGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDIDGKTYLN 96

WLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFT LKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

18 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSHYAMTWV 143

Chain RQAPGKGLEWVSTITTGGSHTYYADSVDGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

LightChain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDIDGKTYLN 96

WLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFT LKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

19 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSQYAMNW 144

Chain VRQAPGKGLEWVSTITKKGSFTYYADSVDGRFTISRD

NAKNSLYLQMNSLRAEDTAVYYCARQDGEFLMDYW GQGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDSDGKTYL 2015

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF 26555_(22)

TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSQYAMNW 145

Chain VRQAPGKGLEWVSTIKKGGSFTYYADSVDGRFTISRD

NAKNSLYLQMNSLRAEDTAVYYCARQDGEFLMDYW GQGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDSDGKTYL 2015

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF 26555_(22)

TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

21 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 146

Chain RQAPGKGLEWVSTITKGGSYTYYADSVDGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

LightChain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDSDGKTYL 2015

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF 26555_(22)

TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

22 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 109

Chain RQAPGKGLEWVSTITTGGSHTYYADSVEGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGHFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDVDGKTYL 147

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTYFPFTFGQGTKVEIKR

23 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSHYAMNW 148

Chain VRQAPGKGLEWVSTITTGGSYTYYADSVEGRFTISRD

NAKNSLYLQMNSLRAEDTAVYYCARQDGHFLMDYW GQGTLVTVSS

LightChain DVVMTQTPLSLPVTLGQPASISCKSSPSLLDIDGKTYLN 108

WLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFT LKISRVEAEDVGVYYCWQGTHLPFTFGQGTKVEIKR

Table 20 Clones Variable CDR1 Amino SEQ CDR2 Amino SEQ CDR3 Amino SEQ

regions acid sequences ID acid sequences ID acid sequences ID

NOS: NOS: NOS:

1001 Heavy SYAMN 149 TITTGGSYTYY 150 QDGNFLMDY 151

Chain ADSVDG

Light KSSQSLLDID 152 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

1015 Heavy SYAMN 149 TITTGGSHTYY 155 QDGNFLMDY 151

Chain ADSVEG

Light KSSQSLLDID 152 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

1021 Heavy SYAMN 149 TITTGGSYTYY 150 QDGHFLMDY 156

Chain ADSVDG

Light KSSQSLLDID 152 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

1023 Heavy SYAMN 149 TITTGGSYTYY 150 QDGNFLMDY 151

Chain ADSVDG

Light KSSQSLLDID 152 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

1024 Heavy SYAMS 157 TITTGGSYTYY 150 QDGNFLMDY 151

Chain ADSVDG

Light KSSQSLLDLD 158 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

1104 Heavy SYAMN 149 TITTGGSHTYY 159 QDGNFLMDY 151

Chain ADSVQG

Light KSSQSLLDVD 160 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

1123 Heavy SYAMN 149 TITTGGSHTYY 155 QDGHFLMDY 156

Chain ADSVEG

Light KSSQSLLDID 152 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

1202 Heavy SYAMN 149 TITTGGSYTYY 150 QDGNFLMDY 151

Chain ADSVDG

Light KSSQSLLDID 152 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

1208 Heavy SYAMN 149 TITTGGSHTYY 155 QDGHFLMDY 156

Chain ADSVEG

Light KSSQSLLDVD 160 LVSKLDS 153 WQGTYLPF 161

Chain GKTYLN

1214 Heavy SYAMN 149 TITTGGSYTYY 162 QDGNFLMDY 151

Chain ADSVEG

Light KSSQSLLDVD 160 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

1216 Heavy HYAMN 163 TITTGGSYTYY 162 QDGHFLMDY 156

Chain ADSVEG

Light KSSQSLLDID 152 LVSKLDS 153 WQGTHLPF 164

Chain GKTYL

1223 Heavy SYAMN 149 TITTGGSYTYY 162 QDGHFLMDY 156

Chain ADSVEG

Light KSSQSLLDID 152 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

1224 Heavy SYAMS 157 TITTGGSYTYY 150 QDGNFLMDY 151

Chain ADSVDG

Light KSSQSLLDID 152 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

1232 Heavy SYAMN 149 TITTGGSYTYY 150 QDGHFLMDY 156

Chain ADSVDG

Light KSSQSLLDVD 160 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

1234 Heavy SYAMN 149 TITTGGSYTYY 165 QDGNFLMDY 151

Chain ADSVQG

Light KSSQSLLDID 152 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

1238 Heavy SYAMN 149 TITTGGSYTYY 162 QDGHFLMDY 156

Chain ADSVEG

Light KSSQSLLDVD 160 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

1243 Heavy SYAMN 149 TITTGGSHTYY 166 QDGHFLMDY 156

Chain ADSVHG

Light KSSQSLLDID 152 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

1248 Heavy SYAMN 149 TITTGGSHTYY 167 QDGHFLMDY 156

Chain ADSVDG

Light KSSQSLLDID 152 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

3007 Heavy SYAMN 149 TITLGGSYTYY 168 QDGNFLMDY 151

Chain ADSVQG

Light KSSQSLLDLD 158 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

3016 Heavy SYAMN 149 TITTGGSHTYY 155 QDGNFLMDY 151

Chain ADSVEG

Light KSSQSLLDSD 169 LVSKLDS 153 WQGTHLPF 164

Chain GKTYLN

3024 Heavy SYAMS 157 TITTGGSYTYY 165 QDGNFLMDY 151

Chain ADSVQG

Light KSSQSLLDSD 169 LVSKLDS 153 WQGTHLPF 164

Chain GKTYLN

3115 Heavy SYAMN 149 TITTGGSHTYY 167 QDGNFLMDY 151

Chain ADSVDG

Light KSSQSLLDVD 160 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

3120 Heavy SYAMN 149 TITTGGSYTYY 162 QDGYFLMDY 170

Chain ADSVEG

Light KSSQSLLDID 152 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

3131 Heavy SYAMS 157 TITTGGSYTYY 165 QDGQFLMDY 171

Chain ADSVQG

Light KSSQSLLDID 152 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

3203 Heavy SYAMS 157 TITTGGSYTYY 162 QDGNFLMDY 151

Chain ADSVEG

Light KSSQSLLDLD 158 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

3241 Heavy SYAMN 149 TITTGGSYTYY 162 QDGNFLMDY 151

Chain ADSVEG

Light KSSQSLLDID 152 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

4011 Heavy SYAMN 149 TITTGGSHTYY 155 QDGNFLMDY 151

Chain ADSVEG

Light KSSQSLLDVD 160 LVSKLDS 153 WQGTHLPF 164

Chain GKTYLN

4017 Heavy SYAMN 149 TITTGGSYTYY 165 QDGYFLMDY 131

Chain ADSVQG

Light KSSQSLLDID 152 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

4034 Heavy SYAMN 149 TITTGGSHTYY 155 QDGYFLMDY 131

Chain ADSVEG

Light KSSQSLLDID 152 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

4041 Heavy SYAMS 157 TITTGGSHTYY 155 QDGYFLMDY 131

Chain ADSVEG

Light KSSQSLLDVD 160 LVSKLDS 153 WQGTHFPF 154

Chain GKTYL

4141 Heavy SYAMN 149 TITTGGSHTYY 155 QDGYFLMDY 131

Chain ADSVEG

Light KSSQSLLDVD 160 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

4146 Heavy SYAMN 149 TITTGGSYTYY 162 QDGYFLMDY 131

Chain ADSVEG

Light KSSQSLLDID 152 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

4206 Heavy SYAMN 149 TITTGGSHTYY 155 QDGNFLMDY 151

Chain ADSVEG

Light KSSQSLLDVD 160 LVSKLDS 153 WQGTHLPF 164

Chain GKTYLN

4208 Heavy SYAMS 157 TITTGGSYTYY 165 QDGNFLMDY 151

Chain ADSVQG

Light KSSQSLLDTD 171 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

4278 Heavy KYAMN 172 TITLGGSYTYY 173 QYLDGNFLM 174

Chain ADSVDG DY

Light KSSQSLLDID 152 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

4287 Heavy KYAMN 172 TITTGGSYTYY 162 QHPYGNFLM 175

Chain ADSVEG DY

Light KSSQSLLDID 152 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

1 Heavy SYAMN 149 TITTGGSYTYY 150 QDGNFLMDY 151

Chain ADSVDG

Light KSSPSLLDSD 176 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

2 Heavy SYAMN 149 TITTGGSYTYY 162 QDGNFLMDY 151

Chain ADSVEG

Light KSSPSLLDID 177 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

3 Heavy SYAMN 149 TITTGGSHTYY 167 QDGNFLMDY 151

Chain ADSVDG

Light KSSPSLLDVD 178 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

4 Heavy SYAMN 149 TITTGGSHTYY 155 QDGNFLMDY 151

Chain ADSVEG

Light KSSPSLLDVD 178 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

Heavy SYAMN 149 TITTGGSYTYY 150 QDGNFLMDY 151

Chain ADSVDG

Light KSSPSLLDSD 176 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

6 Heavy SYAMN 149 TITTGGSYTYY 162 QDGNFLMDY 151

Chain ADSVEG

Light KSSPSLLDSD 176 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

7 Heavy SYAMN 149 TITTGGSYTYY 150 QDGNFLMDY 151

Chain ADSVDG

Light KSSPSLLDVD 178 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

8 Heavy SYAMN 149 TITTGGSYTYY 162 QDGNFLMDY 151

Chain ADSVEG

Light KSSPSLLDVD 178 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

9 Heavy SYAMN 149 TITTGGSYTYY 165 QDGNFLMDY 151

Chain ADSVQG

Light KSSPSLLDVD 178 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

Heavy SYAMN 149 TITTGGSYTYY 165 QDGNFLMDY 151

Chain ADSVQG

Light KSSPSLLDVD 178 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

11 Heavy SYAMN 149 TITTGGSYTYY 162 QDGNFLMDY 151

Chain ADSVEG

Light KSSPSLLDID 177 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

12 Heavy SYAMN 149 TITTGGSHTYY 155 QDGHFLMDY 156

Chain ADSVEG

Light KSSPSLLDID 177 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

13 Heavy SYAMN 149 TITTGGSHTYY 155 QDGNFLMDY 151

Chain ADSVEG

Light KSSPSLLDID 177 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

14 Heavy KYAMN 172 TITLGGSYTYY 173 QYLDGNFLM 174

Chain ADSVDG DY

Light KSSPSLLDSD 176 LVSKLDS 153 WQGTYFPF 179

Chain GKTYLN

Heavy KYAMN 172 TITTGGSYTYY 162 QHPYGNFLM 175

Chain ADSVEG DY

Light KSSPSLLDSD 176 LVSKLDS 153 WQGFYFPF 180

Chain GKTYLN

16 Heavy HYAMT 181 TITTGGSHTYY 155 QDGNFLMDY 151

Chain ADSVEG

Light KSSPSLLDID 177 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

17 Heavy SYAMT 182 TITTGGSHTYY 159 QDGNFLMDY 151

Chain ADSVQG

Light KSSPSLLDID 177 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

18 Heavy HYAMT 181 TITTGGSHTYY 167 QDGNFLMDY 151

Chain ADSVDG

Light KSSPSLLDID 177 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

19 Heavy QYAMN 183 TITKKGSFTYY 184 QDGEFLMDY 185

Chain ADSVDG

Light KSSPSLLDSD 176 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

Heavy QYAMN 183 TIKI(GGSFTYY 186 QDGEFLMDY 185

Chain ADSVDG

Light KSSPSLLDSD 176 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

21 Heavy SYAMN 149 TITKGGSYTYY 187 QDGNFLMDY 151

Chain ADSVDG

Light KSSPSLLDSD 176 LVSKLDS 153 WQGTHFPF 154

Chain GKTYLN

22 Heavy SYAMN 149 TITTGGSHTYY 155 QDGHFLMDY 156

Chain ADSVEG

Light KSSPSLLDVD 178 LVSKLDS 153 WQGTYFPF 179

Chain GKTYLN

23 Heavy HYAMN 188 TITTGGSYTYY 162 QDGHFLMDY 156

Chain ADSVEG

Light KSSPSLLDID 177 LVSKLDS 153 WQGTHLPF 164

Chain GKTYLN

11-4: Cloning of IgG Gene of Clone 308-4 Antibody Mutant That Is Anti-TFPI Antibody

Obtained by Yeast Display

The light-chain variable region gene of the 308-4 antibody mutant that is the

anti-TFPI antibody obtained in Examples 11-2 and 11-3 were subjected to PCR using

PrimeSTAR HS DNA polymerase (CAT.NO.R40B, Takara) together with a KpnI

containing forward primer (Table 21; SEQ ID NO: 189) and a reverse primer (Table 21;

SEQ ID NO: 190). In addition, the kappa constant light region of the human antibody

was subjected to PCR using a forward primer (Table 21; SEQ ID NO: 191) and a reverse

primer (Table 21; SEQ ID NO: 192). The PCR was performed under the following

conditions: 10 min at 94°C; and then 30 cycles, each consisting of 15 see at 94°C, 30 see

at 56°C and 90 see at 72°C; and then 10 min at 72°C. The amplified genes were

electrophoresed on 1% agarose gel to confirm the DNA bands having the expected size,

and were isolated using a gel extraction kit. Next, the light-chain variable region and the

light-chain constant region were mixed with each other at a ratio of 1:1, and the mixture

was subjected to overlapping PCR using a forward primer (Table 20; SEQ ID NO: 189)

and a reverse primer (Table 20; SEQ ID NO: 192) under the following conditions: 10 min

at 94°C; and then 30 cycles, each consisting of 15 see at 94°C, 30 see at 56°C and 90 see

at 72°C; followed by 10 min at 72°C. The amplified gene was electrophoresed on 1%

agarose gel to confirm the DNA band having the expected size, and was isolated using a

gel extraction kit. The isolated gene was treated with a KpnI (CAT.NO.R142L,NEB)

and HindIl (CAT.NO.RO104L, NEB) restriction enzymes at 37°C for 12 hours, and then

separated on 1% agarose gel. A pcIW plasmid vector was digested in the same manner

and separated on agarose gel. Using T4 DNA ligase (Cat.No.M0203S, NEB), the

isolated light-chain region gene was ligated into the NotI and HindIl sites of a linear

pcIW vector. The ligation product was transformed into XL1-Blue bacteria

were plated on a carbenicillin-containing LB plate (Cat.No.LN004CA, NaraeBiotech)

and cultured at 37°C for 12 hours or more, and single colonies were selected from the

plate and cultured. Next, a plasmid was isolated from the cells using a plasmid mini-kit

(Cat.No.27405, QIAGEN) and identified by DNA sequencing.

The heavy-chain variable region was subjected to PCR using the heavy-chain

variable region gene of the 308-4 antibody mutant as a template and PrimeSTAR HS

DNA polymerase (Takara) together with a KpnI-containing reverse primer (Table 21;

SEQ ID NO: 193) and an ApaI-containing reverse primer (Table 21; SEQ ID NO: 194).

The PCR was performed under the following conditions: 2 min at 98°C; and then 30

cycles, each consisting of 10 see at 98°C, 10 see at 58°C and 30 see at 72°C; followed by

5 min at 72°C. The amplified gene was electrophoresed on 1% agarose gel to confirm the

DNA band having the expected size, and was isolated using a gel extraction kit. Next, the

three isolated genes were treated with KpnI and Apal restriction enzymes at 37°C for 4

hours. The gene treated with the restriction enzymes was separated on 1% agarose gel.

A pCIW plasmid vector was also digested in the same manner and separated on agarose

gel. Using T4 DNA ligase, the separated gene was ligated into the KpnI (CAT.

NO.R0142L, NEB) and Apal (CAT.NO.R114L, NEB) sites of a linear pclw vector

containing the human heavy-chain constant region. The ligation product was transformed

into XL1-Blue bacteria (Electroporation-Competent Cells; Stratagene), and the bacterial

cells were plated on a carbenicillin-containing LB plate (Cat.No.LN004CA,

selected from the plate and cultured. Then, a plasmid was isolated from the cells using a

plasmid mini-kit (Cat.No.27405, QIAGEN), and DNA sequencing of the isolated plasmid

was performed.

Table 21 below shows the primers used in IgG gene cloning of the clone 308-4

antibody mutant that is the anti-TFPI antibody obtained by yeast display.

Table 21 Names Nucleic acid sequences SEQ ID

NOS:

VH Fo TGCTGTGGGTGAGTGGTACCTGTGGG GAA GTC CAG 189

CTG GTG GAG TCT GGA GGT

VH Re AGT GGG AAC ACG GAG GGC CCC TTG GTG CTG 190

GCGGAGCTCACAGTCACCAGCGTGCC

VL Fo TGCTGTGGGTGAGTGGTACCTGTGGG GAC GTG GTG 191

ATG ACA CAG ACG CCG CTG

VL ReCL overlap GAT GAA CAC AGA AGG GGC AGC CAC CGT GCG 192

TTT AAT TTC AAC CTT AGT GCC TTG GCC GAA CGT AAA

Ck Fo ACG GTG GCT GCC CCT TCT GTG TTC ATC 193

Ck Re GAT TGG ATC CAA GCT TAC TAG CAC TCA CCC CTG 194

TTG AAA GAC TTA

11-5: Production and Purification of Anti-TFPI 308-4 Clone Antibody Mutant IgG

In order to produce and purify the anti-TFPI clone antibody mutant cloned in

Example 11-4, Expi293FTM cells were seeded at a concentration of 2.5 X 106 cells/mL on

one day before transfection. After 24 hours of culture (37°C, 8% C02, 125 rpm),

Expi293 T M Expression medium (Cat.No.A1435101, Gibco) was added to prepare 30 mL

of the cells at a concentration of 2.5 X 106 cells/mL (viability > 95%). 30 g of DNA

(pclw-anti-TFPI heavy chain: 15[g, pclw-anti-TFPI light chain: 15g) was diluted in

OptiProTMSEM medium (Cat.No.12309019, Gibco) to a total volume of 1.5 mL and

incubated at room temperature for 5 minutes. 80 L of ExpiFectamineTM293 reagent

(Cat.No.A14524, Gibco) was added to 1.5 mL of OptiProTMSEM medium

of suspension culture (37°C, 8% C02, 125 rpm), 150 L of ExpiFectamine TM 293

Enhancer 1 (Cat.No.A14524, Gibco) and 1.5 mL of ExpiFectamine TM 293 Enhancer 2

100 pL of the protein A resin MabSelect Xtra (Cat.No.17-5269-02, GE Healthcare) was

column-snap cap (Cat.No.69725, Thermo), and extracted using the QIAvac 24 Plus

(Cat.No.19413, QIAGEN) vacuum manifold so that only the resin remained in the

column. The resin was washed with 5 mL of protein A binding buffer, and then

was incubated at room temperature for 2 minutes and eluted by centrifugation at 1000 rpm for 1 minute. The eluate was neutralized by addition of 2.5 L of 1.5M Tris-HCl

Nanodrop 200C (Thermo Scientific). Fractions having the protein detected therein were

collected, and the buffer was replaced with PBS (phosphate-buffered saline) buffer using

5 mL of 7K MWCO (Cat.No.0089892, Pierce) in Zeba Spin Desalting Columns. Next,

electrophoresis (SDS-PAGE) of the protein was performed under reducing and non

state of the antibody, and the antibody was stored at 4°C.

As a result, protein electrophoresis (SDS-PAGE) indicated that the anti-TFPI

308-4 clone antibody mutant was purified in a good state.

Example 12: Preparation of Anti- TFPI 308-4 Clone Affinity-Matured Antibody

Using Phage Display Fab Library

12-1: Construction of Phage Display Fab Library

In order to construct an Fab library, a heavy-chain variable region library was

constructed, and then a light-chain variable region library was constructed. Specifically,

for the PCR of heavy-chain variable region fragment 1, the heavy-chain variable region

gene sequence of the anti-TFPI 308-4 clone was used as a template together with a

forward primer (Table 18; SEQ ID NO: 40) and a reverse primer (Table 18; SEQ ID

NOs: 41 to 48); for the PCR of heavy-chain variable region fragment 2, the heavy-chain

variable region gene sequence of the anti-TFPI 308-4 clone was used as a template

together with a forward primer (Table 18; SEQ ID NO: 49) and a reverse primer (Table

18; SEQ ID NOs: 50 to 61); and for the PCR of heavy-chain variable region fragment 2,

the heavy-chain variable region gene sequence of the anti-TFPI 308-4 clone was used as

a template together with a forward primer (Table 18; SEQ ID NO: 62) and a reverse

primer (Table 18; SEQ ID NOs: 63 to 69). The PCR of each of the fragments was performed using AccuPower Pfu PCR PreMix (CAT.NO.K-2015, Bioneer) under the following conditions: 2 min at 95°C; and then 30 cycles, each consisting of 30 see at

95°C, 30 see at 55°C and 60 see at 72°C; followed by 10 min at 72°C. The amplified

genes were electrophoresed on 1% agarose gel to confirm the DNA bands having the

expected sizes, and was isolated using a gel extraction kit (QAquick Gel Extraction Kit,

QIAGEN). The isolated heavy-chain variable region fragment genes were adjusted to a molar ratio of 1:1:1 and used as a template together with a forward primer (Table 18;

the amplified genes was performed using Takara primer star PCR premix (Takara) under

the following conditions: 2 min at 95°C; and then 20 cycles, each consisting of 10 see at

95°C, 20 see at 55°C and 30 see at 72°C; followed by 5 min at 72°C. The amplified gene

thereby obtaining a heavy-chain variable region gene. The obtained gene was treated

with XhoI (CAT.No.R0146L, NEB) and Apal (Cat.No.RO114L, NEB) restriction

enzymes at 37°C for 4 hours. The gene was separated on 1% agarose gel. Using T4

DNA ligase (Cat.No.M0203S, NEB), the separated gene was ligated into the XhoI and

Apal sites of a linear pComb3x vector containing the 308-4 light-chain variable-constant

regions. The ligation product was transformed into XL1-Blue bacteria (Electroporation

competent cells; Cat.No.200228, Stratagene), and then the bacterial cells were cultured in

300 ml of LB medium at 37°C at 220 rpm for 1 hour, and then treated with 150 L of

Carbenicillin and 300 L of tetracycline, followed by suspension culture at 37°C at 220

rpm for 12 hours or more. Next, the constructed heavy-chain variable region library

plasmid was isolated using a Midi prep kit (CAT.No.12143, QIAGEN). To determine

the size of the library, 100 p2 of the culture medium was collected at 1 hour after transformation, and plated on a Carbenicillin-containing LB plate (Cat.No.LN004CA,

NaraeBiotech) by a serial dilution method, after which it was incubated at 37°C for 12

hours or more, and then subjected to colony counting.

For the PCR of light-chain variable region fragment 1, the light-chain variable

forward primer (Table 18; SEQ ID NO: 72) and a reverse primer (Table 18; SEQ ID

NOs: 73 to 82); and for the PCR of light-chain variable region fragment 2, the light-chain

together with a forward primer (Table 18; SEQ ID NO: 83) and a reverse primer (Table

18; SEQ ID NOs: 84 to 87). The PCR of each of the fragments was performed using

AccuPower Pfu PCR PreMix (CAT. NO. K-2015, Bioneer) under the following

conditions: 2 min at 95°C; and then 30 cycles, each consisting of 30 see at 95°C, 30 see at

55°C and 60 see at 72°C; followed by 10 min at 72°C. The amplified genes were

electrophoresed on 1% agarose gel to confirm the DNA bands having the expected sizes,

and were isolated using a gel extraction kit (QAquick Gel Extraction Kit, QIAGEN).

The light-chain variable region fragment genes were adjusted to a molar ratio of 1:1 and

used as a template together with a forward primer (Table 18; SEQ ID NO: 91) and a

reverse primer (Table 18; SEQ ID NO: 92) in PCR. The PCR of the amplified genes was

performed using Takara primer star PCR premix (Takara) under the following conditions:

2 min at 95°C; and then 20 cycles, each consisting of 10 see at 95°C, 30 see at 55°C and

40 see at 72°C; followed by 5 min at 72°C. The amplified gene was electrophoresed on

1% agarose gel to confirm the DNA band having the expected size, and was isolated

using a gel extraction kit (QAquick Gel Extraction Kit, QIAGEN), thereby obtaining a

light-chain variable region gene. The obtained gene was treated with NruI

(CAT.No.R0192L, NEB) and XbaI (Cat.No.R0145L, NEB) restriction enzymes at 37°C for 4 hours. The gene treated with the restriction enzymes was separated on 1% agarose gel. Using T4 DNA ligase (Cat.No.M0203S, NEB), the separated gene was ligated into the NruI and XbaI sites of a linear pComb3x library containing the 308-4 heavy-chain variable region library. The ligation product was transformed into XL1-Blue bacteria

(Electroporation-competent cells; Cat.No.200228, Stratagene), and then the bacterial cells

were cultured in 300 ml of LB medium at 37°C at 220 rpm for 1 hour, and then treated

with 150 L of carbencillin and 300 L of tetracycline, followed by shake culture at 37°C

at 220 rpm for 1 hour. Next, the cells were treated with 4.5 mL (10"pfu) of VCS M13

helper phage, and then shake-cultured at 37°C at 220 rpm for 1 hour. Next, the cells were

treated with 300 L of kanamycin and 300 L of carbenicillin and cultured overnight at

37°C at 220 rpm. On the next day, the cultured cells were centrifuged at 4000 rpm for 20

minutes, and the supernatant was transferred onto a fresh container. To precipitate the

phage, 5X PEG/NaCl was added to the supernatant at IX, and then allowed to stand on

ice for 30 minutes or more. The precipitated phage was centrifuged at 8000 rpm for 30

minutes. The supernatant was discarded, and the precipitated phage was resuspended in

10 mL of PBS. To remove cell debris, the phage suspended in 10 mL of PBS was

centrifuged at 14,000 rpm for 10 minutes, and the supernatant was isolated and stored at

4°C. To determine the size of the library, 100 p2 of the culture medium was collected at

1 hour after transformation, and plated on a Carbenicillin-containing LB plate

(NaraeBiotech) by a serial dilution method, after which it was incubated at 37°C for 12

hours or more, and then subjected to colony counting.

12-2: Selection of Anti-TFPI Antibody Mutant

1 mL of the human recombinant protein TFPI was added to a solid phase

polystyrene tube (Cat.No.444202, Nunc) at a concentration of 1 g/m, and the tube was

coated with the protein at 4°C for 12 hours or more and washed three times with 5 mL of

0.05% PBST. The TFPI-coated Immuno tube was blocked with 5 mL of1%BSA/PBS at

room temperature for 2 hours. The blocking buffer was removed from the Immuno tube,

and then the tube was treated with the phage library and incubated at room temperature

for 2 hours. Next, the tube was washed four times with 5 mL of PBST. The Immuno tube

was treated with 1 mL glycine (pH 2.0) elution buffer and incubated at room temperature

for 10 minutes, and the eluted phage of the supernatant was neutralized by addition of

100 p2 of 1.5M Tris-Cl (pH 8.8). 10 mL of XLI-Blue electroporation-competent cells

(OD600 = 0.8-1.0) cultured for about 2 hours were treated with the neutralized phage.

After infection at room temperature for 30 minutes, 10 mL of SB, 20 pe of tetracycline

(50 mg/mL) and 10 p2 of carbenicillin (100 mg/mL) were added to 10 mL of the infected

XLI-Blue electroporation-competent cells which were then shake-cultured at 200 rpm at

37°C for 1 hour. Then, the cells were treated with 1 mL of VCSM13 helper phage (>

10 11pfu/mL) and shake-cultured at 200 rpm at 37°C for 1 hour. After 1 hour of culture,

the cells were treated with 80 mL of SB, 100 p2 of kanamycin and 100 p2 of carbenicillin

(100 mg/mL) and cultured overnight at 37°C at 200 rpm. The library cultured for 12

hours or more was centrifuged at 4000 rpm for 15 minutes to isolate the supernatant, and

5X PEG/NaCl buffer was added to the supernatant at IX, and then allowed to stand on

ice for 30 minutes. The supernatant was removed by centrifugation at 8000 rpm for 30

minutes. The pellets were resuspended in 2 mL of 1% BSA/PBS, and then centrifuged at

12000 rpm for 10 minutes, and the supernatant was collected and used in the next

panning. The above-described procedure was repeated four times.

12-3: Preparation of Anti-TFPI Individual Clone Antibodies by ELISA

Single colonies were collected from the finally amplified library, and then

cultured in 1.5 mL of SB/carbenicillin at 37C at 220 rpm until an OD600 of about 0.8

1.0 was reached, followed by incubation with 1 mM IPTG at 30°C at 200 rpm for 12

hours. Next, the cells were centrifuged at 5500 rpm for 5 minutes, and the supernatant

was added to a TFPI antigen-coated ELISA plate, incubated at room temperature for 2

hours, and then washed four times with PBST (1XPBS, 0.05% tween 20). Next, a 1:5000

dilution of an HRP/anti-hFab-HRP conjugate (CAT.No.A0293, Sigma) with 1%

BSA/1XPBS was added to the cells, after which the cells were incubated at room

temperature for 1 hour and washed four times with PBST (1XPBS, 0.05% tween 20).

Then, the cells treated with a TMB solution for 5-10 minutes, and a TMB stop solution

was added to the cells. Next, the absorbance at a wavelength of 450 nm was measured

using the TECAN sunrise, and clones having high O.D values were selected as individual

clones.

As a result, as shown in Table 23 below, clones that bind specifically to human

application)'.

Table 23 below 8 shows the CDR amino acid sequences of the clone antibodies

of Table 22, identified based on the Kabat numbering system.

Table 22 Clones Variable Amino Acid Sequences SEQ ID

Regions NOS:

A24 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFHSYAMNW 195

Chain VRQAPGKGLEWVSTITTRGSYTYYADSVEGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDVDGKTYL 103

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

A25 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 97

Chain RQAPGKGLEWVSTITTGGSHTYYADSVEGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

LightChain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDRDGKTYL 196

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

A52 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 197

Chain RQAPGKGLEWVSTITTGGSHTYYADSVDGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

LightChain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDIDGKTYL 96

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

A63 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 198

Chain RQAPGKGLEWVSTITTGGSYTYYADSVQGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

LightChain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDVDGKTYL 103

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

A67 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 2015

Chain RQAPGKGLEWVSTITTGGSYTYYADSVEGRFTISRDN 26555_(25

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG) QGTLVTVSS

LightChain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDLDGKTYL 101

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

A71 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMNWV 199

Chain RQAPGKGLEWVSTITTGGSYTYYADSVHGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

LightChain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDTDGKTYL 130

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

A74 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMHWV 200

Chain RQAPGKGLEWVSTITTGGSYTYYADSVQGRFTISRDN

AKNSLYLQMNSLRAEDTAVYYCARQDGNFLMDYWG QGTLVTVSS

Light Chain DVVMTQTPLSLPVTLGQPASISCKSSQSLLDIDGKTYL 96

NWLQQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDF TLKISRVEAEDVGVYYCWQGTHFPFTFGQGTKVEIKR

Table 23 Clones Variable CDR1AA SEQ CDR2 AA SEQ CDR3 AA SEQ

Regions sequences ID sequences ID sequences ID

NOS: NOS: NOS:

A24 Heavy SYAMN 149 TITTRGSYTY 200 QDGNFLMDY 151

Chain YADSVEG

Light Chain KSSQSLLD 160 LVSKLDS 153 WQGTHFPF 154

VDGKTYLN

A25 Heavy SYAMN 149 TITTGGSHTY 155 QDGNFLMDY 151

Chain YADSVEG

Light Chain KSSQSLLDR 201 LVSKLDS 153 WQGTHFPF 154

DGKTYLN

A52 Heavy SYAMN 149 TITTGGSHTY 167 QDGNFLMDY 151

Chain YADSVDG

Light Chain KSSQSLLDI 152 LVSKLDS 153 WQGTHFPF 154

DGKTYLN

A63 Heavy SYAMN 149 TITTGGSYTY 165 QDGNFLMDY 151

Chain YADSVQG

Light Chain KSSQSLLD 160 LVSKLDS 153 WQGTHFPF 154

VDGKTYLN

A67 Heavy SYAMN 149 TITTGGSYTY 162 QDGNFLMDY 151

Chain YADSVEG

Light Chain KSSQSLLDL 158 LVSKLDS 153 WQGTHFPF 154

DGKTYLN

A71 Heavy SYAMN 149 TITTGGSYTY 202 QDGNFLMDY 151

Chain YADSVHG

Light Chain KSSQSLLDT 171 LVSKLDS 153 WQGTHFPF 154

DGKTYLN

A74 Heavy SYAMH 203 TITTGGSYTY 165 QDGNFLMDY 151

Chain YADSVQG

Light Chain KSSQSLLDI 152 LVSKLDS 153 WQGTHFPF 154

DGKTYLN

12-4: Cloning of IgG Gene of Anti-TFPI 308-4 Clone Antibody Mutant

Using the obtained anti-TFPI 308-4 clone antibody mutant light-chain variable

region gene as a template, PCR was performed using PrimeSTAR HS DNA polymerase

(Takara) together with a KpnI-containing forward primer (Table 21; SEQ ID NO: 189)

and a reverse primer (Table 21; SEQ ID NO: 190). In addition, using the human

antibody kappa constant light region as a template, PCR was performed with a forward

primer (Table 21; SEQ ID NO: 191) and a reverse primer (Table 21; SEQ ID NO: 192).

The PCR was performed under the following conditions: 10 min at 94°C; and then 30

cycles, each consisting of 15 see at 94°C, 30 see at 56°C and 90 see at 72°C; followed by

10 min at 72°C. The amplified genes were electrophoresed on 1% agarose gel to confirm

the DNA bands having the expected sizes, and were isolated using a gel extraction kit.

Next, the light-chain variable region gene and the light-chain constant region gene were

mixed with each other at a ratio of 1:1, and the mixture was subjected to overlapping

PCR using a forward primer (Table 20; SEQ ID NO: 189) and a reverse primer (Table

20; SEQ ID NO: 192) under the following conditions: 10 min at 94°C; and then 30 cycles,

each consisting of 15 see at 94°C, 30 see at 56°C and 90 see at 72°C; followed by 10 min

at 72°C. The amplified gene was electrophoresed on 1% agarose gel to confirm the DNA

band having the expected size, and was isolated using a gel extraction kit. The isolated

gene was treated with KpnI (CAT.NO.R142L, NEB) and HindIII(CAT.NO.R104L,

NEB) restriction enzymes at 37°C for 12 hours or more, and then separated on 1%

agarose gel. A pcIW plasmid vector was digested in the same manner and separated on

agarose gel. Using T4 DNA ligase (Cat.No.M0203S, NEB), the isolated light-chain

region gene was ligated into the NotI and HindIl sites of a linear pcIW vector. The

ligation product was transformed into XL-Blue bacteria (Electroporation-Competent

Cells; Stratagene, Cat.No.200228), and the bacterial cells were plated on a carbenicillin containing LB plate (Cat.No.LN004CA, NaraeBiotech) and cultured at 37°C for 12 hours or more, and single colonies were selected from the plate and cultured. Next, a plasmid was isolated from the cells using a plasmid mini-kit (Cat.No.27405, QIAGEN) and analyzed by DNA sequencing.

The heavy-chain variable region was subjected to PCR using the heavy-chain

DNA band having the expected size, and was isolated using a gel extraction kit. Next,

the three isolated genes were treated with KpnI and Apal restriction enzymes at 370 C for

4 hours. The gene treated with the restriction enzymes was separated on 1% agarose gel.

gel. Using T4 DNA ligase, the separated gene was ligated into the KpnI (CAT. NO.

R0142L, NEB) and Apal (NEB, CAT. NO. R0114L) sites of a linear pcw vector

into XL1-Blue bacteria (Electroporation-Competent Cells; Stratagene, Cat.No.200228),

and the bacterial cells were plated on a carbenicillin-containing LB plate (NaraeBiotech,

Cat.No.LN004CA) and cultured at 37C for 12 hours or more, and single colonies were

plasmid mini-kit (Cat.No.27405, QIAGEN) and was analyzed by DNA sequencing.

12-5: Production and Purification of Anti-TFPI 308-4 Clone Antibody Mutant IgG

In order to produce and purify the anti-TFPI clone antibody mutant cloned in

Example 12-4, Expi293FTM cells were seeded at a concentration of 2.5 X 106 cells/mL on

OptiProTMSEM medium (Cat.No.12309019, Gibco) to a total volume of 1.5 mL and

incubated at room temperature for 5 minutes. 80 L of ExpiFectamineTM293 reagent

(Cat.No.A14524, Gibco) was added to 1.5 mL of OptiProTMSEM medium

of suspension culture (37°C, 8% C02, 125 rpm), 150 L of ExpiFectamine TM 293

Enhancer 1 (Cat.No.A14524, Gibco) and 1.5 mL of ExpiFectamine TM 293 Enhancer 2

prepared culture medium, followed by rotating incubation at room temperature for 30 minutes. The mixture of the culture medium and the resin was added to the Pierce spin column-snap cap (Cat.No.69725, Thermo), and extracted using the QIAvac 24 Plus

(Cat.No.19413, QIAGEN) vacuum manifold so that only the resin remained in the

column. The resin was washed with 5 mL of protein A binding buffer, and then

5 mL of 7K MWCO (Cat.No.0089892, Pierce) in Zeba Spin Desalting Columns. Next,

electrophoresis (SDS-PAGE) of the protein was performed under reducing and non

state of the antibody, and the antibody was stored at 4°C.

As a result, protein electrophoresis (SDS-PAGE) indicated that the anti-TFPI

308-4 clone antibody mutant was purified in a good state.

Example 13: Measurement of Quantitative Affinity of 308-4 Antibody Mutant for

TFPI Antigen

The quantitative affinities of 308-4 clone heavy-chain variable region antibody

mutants 12, 1023, 1202, 3241, which are the anti-TFPI antibodies purified in Examples

11 and 12, for human recombinant TFPI, were measured using a Biacore T-200 (GE

Healthcare) biosensor. Specifically, protein A was immobilized on a CM5 chip (CAT.

No. BR-1005-30, GE Healthcare) to an Rmax of 200 by an amine-carboxyl reaction, and

then each of the purified 12, 1023, 1202 and 3241 clones was bound to the immobilized

protein A. Next, recombinant human TFPI serially diluted in HBS-EP buffer (lOmM

HEPES(pH7.4), 150mM NaCl, 3mM EDTA, 0.005% surfactant P20) was run on the chip

at a concentration of 0.078-5 nM at a flow rate of 30 L/min for 120 seconds for

association and 3600 seconds for dissociation. Dissociation of the TFPI associated with

the antibody was induced by running 10 mM glycine-HCl (pH 1.5) at a flow rate for 30

seconds. The affinities in terms of kinetic rate constants (K. and Kff) and equilibrium

dissociation constant (KD) were evaluated using Biacore T-200 evaluation software, and

the results are shown in Table 24 below.

Table 24 below shows the affinities of the anti-TFPI antibodies for recombinant

human TFPI protein in terms of rate constants (Kon and Kff) and equilibrium dissociation

constant (KD).

Table 24 Kon Koff KD

12 4.87X10 6 3.99X10- 5 8.19X10- 12

1023 4.91X10 6 1.5X10-4 3.01X10-"

1202 7.56X10 6 7.16X10- 5 9.47X10- 12

3241 1.91X10 6 1.4X10-4 7.4X10-"

Example 14: Measurement of Fxa Activity

Blood coagulation is induced by an intrinsic pathway and an extrinsic pathway,

Kunitz 1 (K1), Kunitz 2 (K2) and Kunitz 3 (K3) domains. It is known that the K1

domain binds to FVIIa and the K2 domain binds to FXa. It is known that blood

coagulation is inhibited by the binding between TFPI and the blood clotting factor. Thus, in order to determine the effect of MG1113 (anti-TFPI antibody) on the blood coagulation process, the FXa activity was evaluated.

An assay system was composed only of FXa, TFPI and a candidate antibody so

as to minimize the effects of several factors. When the candidate antibody binds to TFPI,

it does not inhibit the function of FXa, and thus the FXa activity appears. However,

when the candidate antibody does not effectively bind to TFPI, TFPI binds to FXa to

thereby inhibit the function of FXa, and thus the degree of color development decreases.

Thus, the residual activity of FXa which is not inhibited by TFPI is measured by the

degree of substrate degradation. The substrate used herein is the FXa-specific substrate

S-2765, and the substrate is degraded to generate measurable chromophoric pNA at 405

nm. This measurement method is based on an amidolytic assay.

Each of FXa, TFPI, mAb2021 and S-2765 was diluted with assay buffer (20 mM

HEPES, 150 mM NaCl, 1 mg/mL of BSA, 0.02% NaN3, 5 mM CaC2, pH7.4) with

reference to Table 25 below and dispensed in a 1.5 ml tube.

Table 25 Pre-dilution conc. Materials Working conc. (nM) Others (nM)

FXa 2nM 0.5nM

TFPI 40nM 10nM

S-2765 2mM 0.5mM

Standard curve 10nM 0.02, 0.1, 0.5, 2.5nM FXa

mAb2021 160nM 2.5, 5, 10, 20nM Positive Control

50 pL of each of the positive control mAb2021 antibody (anti-TFPI Ab, Novo

Nordisk) and the candidate antibodies was added to each well at concentrations of 20, 10,

5 and 2.5 nM. 50 L of 40 nM TFPI solution was added to each well and allowed to

stand at room temperature for 30 minutes. To obtain a standard curve, 50 pL of FXa

solution was added to each well at varying concentrations, and 50 L of 2 nM FXa

endpoint mode.

As a result, as shown in FIG. 20, the effects of No. 1015, 1021, 1023, 3007, 3016

and 3024 antibodies that are affinity-matured antibodies among the anti-TFPI MG1113

candidate antibodies were analyzed. It was shown that all the antibodies showed

that the TFPI inhibitory effects of the antibodies increase in a concentration-dependent

manner. Among these antibodies, No. 1015 antibody showed the effect of inhibiting

TFPI by about 83% in the sample treated with 20 nM, and the effect of inhibiting TFPI

by about 71% in the sample treated with 10 nM, compared to the positive control sample

not treated with TFPI. In addition, No. 1023 antibody showed the effect of inhibiting

TFPI by about 86% in the sample treated with 20 nM, and the effect of inhibiting TFPI

by about 84% in the sample treated with 10 nM, compared to the positive control sample.

When the effects were compared at a TFPI concentration of 10 nM, it was shown that No.

1023 antibody had a better TFPI inhibitory activity than No. 1015 antibody.

In addition, as shown in FIG. 21, the effects of No. 3036, 3115, 3120, 3131, 4017

and 4141 antibodies that are the affinity-matured antibodies among the anti-TFPI

MG1113 candidate antibodies were analyzed. It was shown that all the antibodies

showed increases in the absorbance in an antibody concentration-dependent manner,

indicating that the TFPI inhibitory effects of the antibodies increase in a concentration dependent manner. Among these antibodies, No. 4017 antibody showed the effect of inhibiting TFPI by about 90% in the sample treated with 20 nM, and the effect of inhibiting TFPI by about 70% in the sample treated with 10 nM, compared to the positive control sample not treated with TFPI.

In addition, as shown in FIG. 22, the effects of No. 1001, 1024, 1104 and 1123

antibodies that are the affinity-matured antibodies among the anti-TFPI MG1113

candidate antibodies were analyzed. It was shown that all the antibodies showed

manner. Among these antibodies, No. 1123 antibody showed the effect of inhibiting

TFPI by about 88% in the sample treated with 20 nM, and the effect of inhibiting TFPI

by about 69% in the sample treated with 10 nM, compared to the positive control sample

not treated with TFPI.

In addition, as shown in FIG. 23, the effects of A24, A25, A51, A52, A63 and

A67 antibodies that are the affinity-matured antibodies among the anti-TFPI MG1113

candidate antibodies were analyzed. It was shown that all the antibodies showed

manner. Among these antibodies, A67 antibody showed the effect of inhibiting TFPI by

about 79% in the sample treated with 20 nM, and the effect of inhibiting TFPI by about

67% in the sample treated with 10 nM, compared to the positive control sample not

treated with TFPI.

In addition, as shown in FIG. 24, the effects of No. 3203, 3241, 4206 and 4208

antibodies that are the affinity-matured antibodies among the anti-TFPI MG1113

candidate antibodies were analyzed. It was shown that all the antibodies showed increases in the absorbance in an antibody concentration-dependent manner, indicating that the TFPI inhibitory effects of the antibodies increase in a concentration-dependent manner. Among these antibodies, No. 3241 antibody showed the effect of inhibiting

TFPI by about 82% in the sample treated with 20 nM, and the effect of inhibiting TFPI

by about 83% in the sample treated with 10 nM, compared to the positive control sample

not treated with TFPI.

In addition, as shown in FIG. 25, the effects of No. 1, 2, 3, 7, 8 and 10 antibodies

that are the affinity-matured antibodies among the anti-TFPI MG1113 candidate

antibodies were analyzed. It was shown that all the antibodies showed increases in the

inhibitory effects of the antibodies increase in a concentration-dependent manner.

Among these antibodies, No. 2 antibody showed the effect of inhibiting TFPI by about

76% in the sample treated with 20 nM, and the effect of inhibiting TFPI by about 79% in

the sample treated with 10 nM, compared to the positive control sample not treated with

TFPI. No. 3 antibody showed the effect of inhibiting TFPI by about 81% in the sample

treated with 20 nM, and the effect of inhibiting TFPI by about 70% in the sample treated

with 10 nM, compared to the positive control sample not treated with TFPI. No. 8

antibody showed the effect of inhibiting TFPI by about 80% in the sample treated with 20

nM, and the effect of inhibiting TFPI by about 69% in the sample treated with 10 nM,

compared to the positive control sample not treated with TFPI.

In addition, as shown in FIG. 26, the effects of No. 1214, 1216, 1224, 1234, 1238

and 4287 antibodies that are the affinity-matured antibodies among the anti-TFPI

MG1113 candidate antibodies were analyzed. It was shown that all the antibodies

indicating that the TFPI inhibitory effects of the antibodies increase in a concentration dependent manner. Among these antibodies, No. 1214 antibody showed the effect of inhibiting TFPI by about 77% in the sample treated with 20 nM, and the effect of inhibiting TFPI by about 63% in the sample treated with 10 nM, compared to the positive control sample not treated with TFPI.

In addition, as shown in FIG. 27, the effects of No. 16, 19, 20, 21 and 23

antibodies that are the affinity-matured antibodies among the anti-TFPI MG1113

candidate antibodies were analyzed. It was shown that all the antibodies showed

manner. Among these antibodies, No. 16 antibody showed the effect of inhibiting TFPI

by about 55% in the sample treated with 20 nM, and the effect of inhibiting TFPI by

about 34% in the sample treated with 10 nM, compared to the positive control sample not

treated with TFPI.

In addition, as shown in FIG. 28, the effects of No. 11, 12, 13 and 1202

antibodies that are the affinity-matured antibodies among the anti-TFPI MG1113

candidate antibodies were analyzed. It was shown that all the antibodies showed

manner. Among these antibodies, No. 11 antibody showed the effect of inhibiting TFPI

by about 89% in the sample treated with 20 nM, and the effect of inhibiting TFPI by

about 81% in the sample treated with 10 nM, compared to the positive control sample not

treated with TFPI. No. 12 antibody showed the effect of inhibiting TFPI by about 82% in

the sample treated with 20 nM, and the effect of inhibiting TFPI by about 82% in the

sample treated with 10 nM, compared to the positive control sample not treated with

TFPI. No. 13 antibody showed the effect of inhibiting TFPI by about 85% in the sample treated with 20 nM, and the effect of inhibiting TFPI by about 76% in the sample treated with 10 nM, compared to the positive control sample not treated with TFPI. No. 1202 antibody showed the effect of inhibiting TFPI by about 87% in the sample treated with 20 nM, and the effect of inhibiting TFPI by about 82% in the sample treated with 10 nM, compared to the positive control sample not treated with TFPI.

Example 15: Measurement of TF/FVIIa/FX Complex

prothrombin into thrombin, which then cleaves fibrinogen into fibrin which acts on blood

coagulation. However, TFPI (tissue factor pathway inhibitor) inhibits the function of

FXa by binding to FXa, thereby interfering with blood coagulation. In order to evaluate

the effect of the anti-TFPI antibody MG1113 in the above-described pathway, a

TF/FVIIa/FXa complex assay was performed. In a state in which TFPI was present

together with or independently of the anti-TFPI antibody MG1113, the extents of

production and inhibition of FXa by a TF/FVIIa complex were measured based on the

extent of color development of a substrate (S2765) degraded by FXa, thereby evaluating

the effect of the anti-TFPI antibody MG1113. In other words, as the TFPI inhibitory

effect of the anti-TFPI antibody MG1113 increases, the production of FXa increases, and

the amount of substrate degraded increases, resulting in an increase in absorbance.

In 1.5 mL tubes, TF (4500L/B, Sekisui diagnostics), FVIIa (Novo Nordisk, Novo

Seven) and FX (PP008A, Hyphen biomed) were diluted with assay buffer (20 mM

HEPES, 150 mM NaCl, 1mg/mL BSA, 0.02% NaN3, 5 mM CaC2, pH 7.4) to the

concentrations shown in Table 26 below, thereby preparing a mixture solution.

Table 26 Material TF FVIIa FX

Concentration 0.6ng/mL lnM 17nM --> 5nM

70 pL of the mixture solution was added to each well of a 96-well plate. To a

blank well, 70 L of assay buffer was added. Each well was incubated at 37°C for 15

minutes, and then 30 L of TFPI was added to each well to a concentration of 50 nM.

control well (a sample not treated with the anti-TFPI antibody MG1113 and TFPI). 30 L

of the anti-TFPI antibody MG1113 was added to each well to concentrations of 12.5, 25,

50 and 100 nM. To each of the blank well, the positive control well (a sample not treated

with the anti-TFPI antibody and TFPI) and the negative control well (a sample not treated

with the anti-TFPI antibody MG1113), 30 L of assay buffer was added, followed by

incubation at 37°C for 15 minutes. 20 L of EDTA (E7889, Sigma-Aldrich) was added to

each well to a concentration of 50 mM. Next, 50 L of S2765 (Chromogenix, S-2765)

was added to each well to a concentration of 200 [M, followed by incubation at 37°C for

10 minutes. Next, the absorbance of each well at 405 nm was measured using a

microplate reader.

Table 27 shows the numerical results obtained by evaluating the effects of the

affinity-matured anti-TFPI MG1113 antibodies by the TF/FVIIa/FX complex assay.

Table 27 Ab Normalized TFPI(5OnM) mAb2021 T417 308-4 1015 1023 4017

Conc.

1OOnM 0.918 0.119 0.937 0.949 0.938 0.944 0.951 0.943

50nM 0.929 0.945 0.926 0.919 0.947 0.919

25nM 0.918 0.873 0.664 0.269 0.795 0.307

12.5nM 0.218 0.242 0.223 0.179 0.228 0.181

6.25nM 0.168 0.179 0.177 0.158 0.168 0.150

3.13nM 0.145 0.147 0.155 0.148 0.152 0.138

1.56nM 0.125 0.134 0.135 0.141 0.143 0.134

Table 28 below shows the numerical results obtained by evaluating the effects of

the affinity-matured anti-TFPI MG1113 antibodies by the TF/FVIIa/FX complex assay.

Table 28 Ab Normalized TFPI(5OnM) mAb2021 T417 308-4 1015 1023 4017

Conc.

1OOnM 100.0% 13.0% 102.0% 103.3% 102.1% 102.8% 103.6% 102.7%

50nM 101.2% 102.9% 100.8% 100.1% 103.1% 100.1%

25nM 100.0% 95.0% 72.3% 29.3% 86.5% 33.4%

12.5nM 23.7% 26.3% 24.2% 19.4% 24.8% 19.7%

6.25nM 18.2% 19.4% 19.2% 17.2% 18.2% 16.3%

3.13nM 15.7% 16.0% 16.8% 16.1% 16.5% 15.0%

1.56nM 13.6% 14.5% 14.7% 15.4% 15.6% 14.5%

As a result, as shown in FIG. 29 and Tables 27 and 28 above, the effects of No.

1015, 1023 and 4017 antibodies that are affinity-matured antibodies among the anti-TFPI

MG1113 candidate antibodies were confirmed. It was shown that all the candidate

antibodies showed increases in the absorbance in an antibody concentration-dependent

manner, indicating that the TFPI inhibitory effects of the antibodies increase in a

concentration-dependent manner. No. 1015 antibody showed the effect of inhibiting

TFPI by 100% in the sample treated with 50 nM, and the effect of inhibiting TFPI by

about 29.3% in the sample treated with 25 nM, compared to the positive control sample

not treated with TFPI. No. 1023 antibody showed the effect of inhibiting TFPI by 100%

in the sample treated with 50 nM, and the effect of inhibiting TFPI by about 86.5% in the

sample treated with 25 nM, compared to the positive control sample. No. 4017 antibody

showed the effect of inhibiting TFPI by 100% in the sample treated with 50 nM, and the

effect of inhibiting TFPI by about 33.4% in the sample treated with 25 nM, compared to

the positive control sample. Thus, it was found that No. 1023 antibody has the high

ability to inhibit TFPI.

Table 29 below shows the numerical results obtained by evaluating the effects of

Table 29 Ab Conc. Normalized TFPI(5OnM) mAb2021 T417 308-4 1023 1123 A67

1OOnM 0.955 0.143 0.966 0.945 0.926 0.935 0.905 0.907

50nM 0.951 0.908 0.909 0.905 0.770 0.895

25nM 0.955 0.880 0.716 0.923 0.272 0.914

12.5nM 0.233 0.251 0.222 0.259 0.157 0.290

6.25nM 0.180 0.186 0.185 0.190 0.150 0.196

3.13nM 0.171 0.160 0.164 0.167 0.151 0.177

1.56nM 0.151 0.145 0.154 0.153 0.140 0.154

Table 30 below shows the numerical results obtained by evaluating the effects of

Table 30 Ab Conc. Normalized TFPI(5OnM) mAb2021 T417 308-4 1023 1123 A67

1OOnM 100.0% 15.0% 101.2% 99.0% 97.0% 98.0% 94.8% 95.0%

50nM 99.6% 95.1% 95.2% 94.8% 80.7% 93.7%

25nM 100.1% 92.2% 75.0% 96.6% 28.5% 95.8%

12.5nM 24.4% 26.3% 23.2% 27.1% 16.4% 30.3%

6.25nM 18.8% 19.5% 19.4% 19.9% 15.7% 20.5%

3.13nM 17.9% 16.8% 17.2% 17.5% 15.8% 18.5%

1.56nM 15.8% 15.2% 16.1% 16.0% 14.7% 16.1%

In addition, as shown in FIG. 30 and Tables 29 and 30 above, No. 1023 antibody

determined to have the highest effect in the above-described assay, together with No.

1123 antibody that is another affinity-matured antibody and the A67 antibody, was

evaluated. It was shown that all the candidate antibodies showed increases in the

inhibitory effects of the antibodies increase in a concentration-dependent manner. No.

1023 antibody showed the effect of inhibiting TFPI by 94.8% in the sample treated with

50 nM, and the effect of inhibiting TFPI by about 96.6% in the sample treated with 25 nM, compared to the positive control sample not treated with TFPI. No. 1123 antibody showed the effect of inhibiting TFPI by 80.7% in the sample treated with 50 nM, and the effect of inhibiting TFPI by about 28.5% in the sample treated with 25 nM, compared to the positive control sample. A67 antibody showed the effect of inhibiting TFPI by 93.7% in the sample treated with 50 nM, and the effect of inhibiting TFPI by about 95.8% in the sample treated with 25 nM, compared to the positive control sample. Thus, it was found that No. 1023 and A67 antibodies are similar to each other in the ability to inhibit TFPI.

Table 31 below shows the numerical results obtained by evaluating the effects of

Table 31 Ab Conc. Normalized TFPI(5OnM) mAb2021 T417 1023 A67 3203 3241

1OOnM 0.915 0.115 0.952 0.931 0.939 0.947 0.932 0.937

50nM 0.953 0.938 0.938 0.938 0.934 0.935

25nM 0.932 0.894 0.914 0.908 0.424 0.911

12.5nM 0.241 0.253 0.290 0.330 0.208 0.305

6.25nM 0.156 0.183 0.185 0.195 0.173 0.197

3.13nM 0.143 0.158 0.160 0.172 0.162 0.171

1.56nM 0.137 0.160 0.135 0.149 0.147 0.157

Table 32 below shows the numerical results obtained by evaluating the effects of

Table 32 Ab Conc. Normalized TFPI(5OnM) mAb2021 T417 1023 A67 3203 3241

1OOnM 100.0% 12.6% 104.0% 101.7% 102.6% 103.5% 101.8% 102.3%

50nM 104.1% 102.5% 102.5% 102.5% 102.1% 102.1%

25nM 101.9% 97.7% 99.8% 99.2% 46.3% 99.6%

12.5nM 26.3% 27.7% 31.6% 36.1% 22.7% 33.3%

6.25nM 17.0% 20.0% 20.2% 21.3% 18.9% 21.5%

3.13nM 15.6% 17.2% 17.5% 18.7% 17.7% 18.7%

1.56nM 15.0% 17.5% 14.7% 16.3% 16.1% 17.2%

In addition, as shown in FIG. 31 and Tables 31 and 32 above, No. 1023 antibody

determined to have the highest effect in the above-described assay, the A67 antibody, and

No. 3203 antibody and No. 3241 antibody which are additional affinity-matured

antibodies, were evaluated. It was shown that all the candidate antibodies showed

manner. No. 1023 antibody showed the effect of inhibiting TFPI by 100% in the sample

treated with 50 nM, and the effect of inhibiting TFPI by about 99.8% in the sample

treated with 25 nM, compared to the positive control sample not treated with TFPI. A67

antibody showed the effect of inhibiting TFPI by 100% in the sample treated with 50 nM,

and the effect of inhibiting TFPI by about 99.2% in the sample treated with 25 nM,

compared to the positive control sample. No. 3203 antibody showed the effect of

inhibiting TFPI by 100% in the sample treated with 50 nM, and the effect of inhibiting

TFPI by about 46.3% in the sample treated with 25 nM, compared to the positive control

sample. No. 3241 antibody showed the effect of inhibiting TFPI by 100% in the sample

treated with 50 nM, and the effect of inhibiting TFPI by about 99.6% in the sample treated with 25 nM, compared to the positive control sample. Thus, it was found that No.

1023, A67 and No. 3241 antibodies are similar to each other in the ability to inhibit TFPI.

Table 33 below shows the numerical results obtained by evaluating the effects of

Table 33 Ab Conc. Normalized TFPI(5OnM) mAb202 T417 1023 2 3 8

1

100nM 0.809 0.04 0.805 0.745 0.810 0.835 0.842 0.834

50nM 0.733 0.509 0.652 0.735 0.743 0.673

25nM 0.344 0.154 0.216 0.416 0.527 0.391

12.5nM 0.082 0.072 0.079 0.083 0.141 0.088

6.25nM 0.050 0.050 0.056 0.052 0.059 0.052

3.13nM 0.047 0.049 0.048 0.045 0.046 0.050

1.56nM 0.043 0.045 0.050 0.045 0.045 0.041

Table 34 below shows the numerical results obtained by evaluating the effects of

Table 34 Ab Conc. Normalized TFPI(50 M) mAb202 T417 1023 2 3 8

1

1OOnM 100.0% 4.9% 99.4% 92.0% 100.1% 103.2% 104.1% 103.0%

50nM 90.6% 62.9% 80.5% 90.9% 91.8% 83.2%

25nM 42.5% 19.0% 26.6% 51.4% 65.1% 48.3%

12.5nM 10.1% 8.9% 9.7% 10.3% 17.4% 10.8%

6.25nM 6.1% 6.2% 6.9% 6.4% 7.2% 6.4%

3.13nM 5.7% 6.0% 5.9% 5.6% 5.7% 6.1%

1.56nM 5.3% 5.5% 6.2% 5.5% 5.6% 5.1%

In addition, as shown in FIG. 32 and Tables 33 and 34 above, the concentration

of FX used for treatment was changed from 17 nM to 5 nM to reduce the reaction rate to

thereby increase resolution for analyzing the effects of the candidate antibodies. No.

1023 antibody selected through the above-described assay, and No. 2, 3 and 8 antibodies

which are additional affinity-matured antibodies, were evaluated. It was observed that

the candidate antibodies showed increases in the absorbance in an antibody

concentration-dependent manner, indicating that the TFPI inhibitory effects of the

antibodies increase in a concentration-dependent manner. It was shown that all the

candidate antibodies showed increases in the absorbance in an antibody concentration

dependent manner, indicating that the TFPI inhibitory effects of the antibodies increase in

a concentration-dependent manner. No. 1023 antibody showed the effect of inhibiting

TFPI by 80.5% in the sample treated with 50 nM, and the effect of inhibiting TFPI by

about 26.6% in the sample treated with 25 nM, compared to the positive control sample

not treated with TFPI. No. 2 antibody showed the effect of inhibiting TFPI by about

99.9% in the sample treated with 50 nM, and the effect of inhibiting TFPI by about

51.4% in the sample treated with 25 nM, compared to the positive control sample. No. 3

antibody showed the effect of inhibiting TFPI by about 91.8% in the sample treated with

50 nM, and the effect of inhibiting TFPI by about 61.5% in the sample treated with 25

nM, compared to the positive control sample. No. 8 antibody showed the effect of inhibiting TFPI by about 83.2% in the sample treated with 50 nM, and the effect of inhibiting TFPI by about 48.3% in the sample treated with 25 nM, compared to the positive control sample.

Table 35 below shows the numerical results obtained by evaluating the effects of

Table 35 Ab Conc. Normalized TFPI(5OnM) mAb202 T417 1023 12 13 1202

1

100nM 0.848 0.035 0.852 0.810 0.846 0.847 0.859 0.859

50nM 0.730 0.600 0.681 0.803 0.818 0.843

25nM 0.462 0.273 0.371 0.489 0.528 0.509

12.5nM 0.105 0.074 0.088 0.097 0.101 0.091

6.25nM 0.062 0.050 0.071 0.067 0.075 0.059

3.13nM 0.046 0.047 0.051 0.048 0.054 0.051

1.56nM 0.044 0.045 0.041 0.041 0.046 0.043

Table 36 below shows the numerical results obtained by evaluating the effects of

Table 36 Ab Conc. Normalized TFPI(5OnM) mAb202 T417 1023 12 13 1202

1

1OOnM 100.0% 4.1% 100.4% 95.5% 99.8% 99.9% 101.3% 101.2%

50nM 86.1% 70.7% 80.3% 94.6% 96.5% 99.4%

25nM 54.4% 32.1% 43.7% 57.7% 62.2% 60.0%

12.5nM 12.4% 8.7% 10.3% 11.4% 11.9% 10.7%

6.25nM 7.3% 5.8% 8.3% 7.8% 8.8% 6.9%

3.13nM 5.4% 5.5% 6.0% 5.6% 6.4% 6.0%

1.56nM 5.2% 5.2% 4.8% 4.8% 5.4% 5.1%

As shown in FIG. 32 and Tables 35 and 36 above, No. 1023 antibody and No. 12,

13 and 1202 antibodies that are additional affinity-matured antibodies were evaluated. It

was observed that the candidate antibodies showed increases in the absorbance in an

the antibodies increase in a concentration-dependent manner. It was shown that all the

TFPI by 80.3% in the sample treated with 50 nM, and the effect of inhibiting TFPI by

about 43.7% in the sample treated with 25 nM, compared to the positive control sample

not treated with TFPI. No. 12 antibody showed the effect of inhibiting TFPI by about

94.6% in the sample treated with 50 nM, and the effect of inhibiting TFPI by about 5 7 .7 % in the sample treated with 25 nM, compared to the positive control sample. No.

13 antibody showed the effect of inhibiting TFPI by about 96.5% in the sample treated

with 50 nM, and the effect of inhibiting TFPI by about 62.2% in the sample treated with

25 nM, compared to the positive control sample. No. 1202 antibody showed the effect of

inhibiting TFPI by about 99.4% in the sample treated with 50 nM, and the effect of

inhibiting TFPI by about 60.0% in the sample treated with 25 nM, compared to the

positive control sample.

Example 16: Measurement of Thrombin Generation

The blood coagulation mechanism is divided into an intrinsic pathway and an

(factor X) and the like. When TF and FVIIa form a complex by an external signal, FX is

activated into FXa. Then, FXa activates prothrombin into thrombin, which then cleaves

fibrinogen into fibrin which acts on blood coagulation. However, TFPI (tissue factor

pathway inhibitor) acts to inhibit the function of FXa by binding to FXa, thereby

interfering with blood coagulation. A thrombin generation assay comprises: treating

plasma with a test sample to be evaluated; and then inspecting the amount of thrombin

produced in the plasma, based on the amount of a fluorescent product produced when the

produced thrombin converts a fluorogenic substrate into the fluorescent product in the

presence of PPP-reagent low; and calibrating the inspected amount of thrombin with the

known amount of thrombin calibrator, thereby measuring the actual generation of

thrombin.

20 L of PPP-reagent low solution was added to the sample loading well of a

prewarmed 96-well plate (round bottom immulon 2HB 96 well plate), and 20 L of

for 10 minutes so that it could bind to TFPI.

80 L of each of the sample dilution (FVIII-deficientplasma) was added to each

of the calibrator and blank wells, and 80 L of the diluted antibody solution was added to each of the remaining wells. A start button at the bottom of the software screen was pressed to execute washing. Washing was performed in a state in which an inlet tube was placed in distilled water in a water bath at 37°C and in which an outlet tube was placed in an empty container. After completion of the washing, the next button was pressed to perform an empty process. The inlet tube was placed in a FluCa solution warmed to

37°C and was primed to fill the tube with the solution. The outlet tube was mounted in

an M hole in a dispenser, and then the next button was pressed to automatically dispense

20 L of FluCa solution into each well, after which a shaking process was performed and

analysis was initiated.

As a result, as shown in FIG. 34, for No. 1023 antibody among the affinity

matured antibodies selected through the Fxa activity assay and the TF/FVIIa/FXa

complex assay, a thrombin generation comparison assay was performed using T417

chimeric antibody. At a concentration of 2.5 nM, the T417 antibody showed an increase

in thrombin peak of about 401%, and No. 1023 antibody showed an increase in thrombin

peak of about 401%, compared to the blank treated with only the sample dilution. In the

case of ETP indicating the total generation of thrombin, in the sample treated with 2.5

nM, the T417 antibody showed an increase in ETP of about 293%, and No. 1023

antibody showed an increase in ETP of about 309%, compared to the negative control

group (having no antibody). When the two antibodies were compared, it was shown that

No. 1023 antibody obtained by affinity maturation has a better effect than the T417

antibody.

Industrial Applicability As described above, the antibody of the present invention, which binds

specifically to TFPI, can activate the extrinsic pathway of blood coagulation by inhibiting

TFPI. Thus, the antibody of the present invention can be effectively used for the

treatment of antibody-induced hemophilia patients and for the prevention of blood

coagulation disease in hemophilia-A or hemophilia-B patients.

Although the present invention has been described in detail with reference to the

specific features, it will be apparent to those skilled in the art that this description is only

for a preferred embodiment and does not limit the scope of the present invention. Thus,

the substantial scope of the present invention will be defined by the appended claims and

equivalents thereof.

SEQUENCE LISTING 26 Feb 2019

<110> MOGAM INSTITUTE FOR BIOMEDICAL RESEARCH

<120> NOVEL ANTIBODY BINDING TO TFPI AND COMPOSITION COMPRISING THE SAME

<130> PF-B1943

<140> PCT/KR2015/014370 <141> 2015-12-29

<150> 10-2015-0026555 2019201141

<151> 2015-02-25

<150> 10-2015-0135761 <151> 2015-09-24

<160> 203

<170> PatentIn version 3.5

<210> 1 <211> 27 <212> DNA <213> Artificial Sequence

<220> <223> VH FR1 Fo

<400> 1 gaagtccagc tggtggagtc tggaggt 27

<210> 2 <211> 57 <212> DNA <213> Artificial Sequence

<220> <223> VH FR1 Re_S

<400> 2 cggggcctga cgaacccagt tcatggcata gctgctgaag gtgaagccgc tcgctgc 57

<210> 3 <211> 57 <212> DNA <213> Artificial Sequence

<220> <223> VH FR1 Re_H

<400> 3 cggggcctga cgaacccagt tcatggcata atggctgaag gtgaagccgc tcgctgc 57

<210> 4 <211> 57 26 Feb 2019

<212> DNA <213> Artificial Sequence

<220> <223> VH FR1 Re_K

<400> 4 cggggcctga cgaacccagt tcatggcata tttgctgaag gtgaagccgc tcgctgc 57 2019201141

<210> 5 <211> 57 <212> DNA <213> Artificial Sequence

<220> <223> VH FR1 Re_R

<400> 5 cggggcctga cgaacccagt tcatggcata tctgctgaag gtgaagccgc tcgctgc 57

<210> 6 <211> 57 <212> DNA <213> Artificial Sequence

<220> <223> VH FR1 Re_T

<400> 6 cggggcctga cgaacccagt tcatggcata agtgctgaag gtgaagccgc tcgctgc 57

<210> 7 <211> 57 <212> DNA <213> Artificial Sequence

<220> <223> VH FR1 Re_Y

<400> 7 cggggcctga cgaacccagt tcatggcata atagctgaag gtgaagccgc tcgctgc 57

<210> 8 <211> 57 <212> DNA <213> Artificial Sequence

<220> <223> VH FR1 Re_I

<400> 8 cggggcctga cgaacccagt tcatggcata aatgctgaag gtgaagccgc tcgctgc 57 26 Feb 2019

<210> 9 <211> 57 <212> DNA <213> Artificial Sequence

<220> <223> VH FR1 Re_L

<400> 9 2019201141

cggggcctga cgaacccagt tcatggcata aaggctgaag gtgaagccgc tcgctgc 57

<210> 10 <211> 27 <212> DNA <213> Artificial Sequence

<220> <223> VH FR2 Fo

<400> 10 tatgccatga actgggttcg tcaggcc 27

<210> 11 <211> 93 <212> DNA <213> Artificial Sequence

<220> <223> VH FR2 Re_T-YH-EQDH

<400> 11 gttatcgcgg gaaatggtga agcgcccttg aacgctatcg gcgtagtagg tgtttgaccc 60

accggttgtg atggtgctga cccattccaa gcc 93

<210> 12 <211> 93 <212> DNA <213> Artificial Sequence

<220> <223> VH FR2 Re_T-RK-EQDH

<400> 12 gttatcgcgg gaaatggtga agcgcccttg aacgctatcg gcgtagtagg ttyttgaccc 60

accggttgtg atggtgctga cccattccaa gcc 93

<210> 13 26 Feb 2019

<211> 93 <212> DNA <213> Artificial Sequence

<220> <223> VH FR2 Re_FYLH-YH-EQDH

<400> 13 gttatcgcgg gaaatggtga agcgcccttg aacgctatcg gcgtagtagg tgtttgaccc 60 2019201141

acctwrtgtg atggtgctga cccattccaa gcc 93

<210> 14 <211> 93 <212> DNA <213> Artificial Sequence

<220> <223> VH FR2 Re_FYLH-RK-EQDH

<400> 14 gttatcgcgg gaaatggtga agcgcccttg aacgctatcg gcgtagtagg ttyttgaccc 60

acctwrtgtg atggtgctga cccattccaa gcc 93

<210> 15 <211> 93 <212> DNA <213> Artificial Sequence

<220> <223> VH FR2 Re_KRI-YH-EQDH

<400> 15 gttatcgcgg gaaatggtga agcgcccttg aacgctatcg gcgtagtagg tgtttgaccc 60

accthttgtg atggtgctga cccattccaa gcc 93

<210> 16 <211> 93 <212> DNA <213> Artificial Sequence

<220> <223> VH FR2 Re_KRI-RK-EQDH

<400> 16 gttatcgcgg gaaatggtga agcgcccttg aacgctatcg gcgtagtagg ttyttgaccc 60 accthttgtg atggtgctga cccattccaa gcc 93 26 Feb 2019

<210> 17 <211> 93 <212> DNA <213> Artificial Sequence

<220> <223> VH FR2 Re_T-YH-EQDH_#2 2019201141

<400> 17 gttatcgcgg gaaatggtga agcgcccnts aacgctatcg gcgtagtagg tatrtgaccc 60

accggttgtg atggtgctga cccattccaa gcc 93

<210> 18 <211> 93 <212> DNA <213> Artificial Sequence

<220> <223> VH FR2 Re_T-RK-EQDH_#2

<400> 18 gttatcgcgg gaaatggtga agcgcccnts aacgctatcg gcgtagtagg ttyttgaccc 60

accggttgtg atggtgctga cccattccaa gcc 93

<210> 19 <211> 93 <212> DNA <213> Artificial Sequence

<220> <223> VH FR2 Re_FYLH-YH-EQDH_#2

<400> 19 gttatcgcgg gaaatggtga agcgcccnts aacgctatcg gcgtagtagg tatrtgaccc 60

acctwrtgtg atggtgctga cccattccaa gcc 93

<210> 20 <211> 93 <212> DNA <213> Artificial Sequence

<220>

<223> VH FR2 Re_FYLH-RK-EQDH_#2 26 Feb 2019

<400> 20 gttatcgcgg gaaatggtga agcgcccnts aacgctatcg gcgtagtagg ttyttgaccc 60

acctwrtgtg atggtgctga cccattccaa gcc 93

<210> 21 <211> 93 2019201141

<212> DNA <213> Artificial Sequence

<220> <223> VH FR2 Re_KRI-YH-EQDH_#2

<400> 21 gttatcgcgg gaaatggtga agcgcccnts aacgctatcg gcgtagtagg tatrtgaccc 60

accthttgtg atggtgctga cccattccaa gcc 93

<210> 22 <211> 93 <212> DNA <213> Artificial Sequence

<220> <223> VH FR2 Re_KRI-RK-EQDH_#2

<400> 22 gttatcgcgg gaaatggtga agcgcccnts aacgctatcg gcgtagtagg ttyttgaccc 60

accthttgtg atggtgctga cccattccaa gcc 93

<210> 23 <211> 27 <212> DNA <213> Artificial Sequence

<220> <223> VH FR3 Fo

<400> 23 gggcgcttca ccatttcccg cgataac 27

<210> 24 <211> 60 <212> DNA

<213> Artificial Sequence 26 Feb 2019

<220> <223> VH FR3 Re_N

<400> 24 gccctggccc caataatcca tcagaaaatt gccatcctgg cgcgcgcaat aatataccgc 60

<210> 25 <211> 60 <212> DNA 2019201141

<213> Artificial Sequence

<220> <223> VH FR3 Re_F

<400> 25 gccctggccc caataatcca tcagaaaaaa gccatcctgg cgcgcgcaat aatataccgc 60

<210> 26 <211> 60 <212> DNA <213> Artificial Sequence

<220> <223> VH FR3 Re_H

<400> 26 gccctggccc caataatcca tcagaaaatg gccatcctgg cgcgcgcaat aatataccgc 60

<210> 27 <211> 60 <212> DNA <213> Artificial Sequence

<220> <223> VH FR3 Re_K

<400> 27 gccctggccc caataatcca tcagaaattt gccatcctgg cgcgcgcaat aatataccgc 60

<210> 28 <211> 60 <212> DNA <213> Artificial Sequence

<220> <223> VH FR3 Re_Q

<400> 28 gccctggccc caataatcca tcagaaattg gccatcctgg cgcgcgcaat aatataccgc 60

<210> 29 26 Feb 2019

<211> 60 <212> DNA <213> Artificial Sequence

<220> <223> VH FR3 Re_R

<400> 29 gccctggccc caataatcca tcagaaatct gccatcctgg cgcgcgcaat aatataccgc 60 2019201141

<210> 30 <211> 60 <212> DNA <213> Artificial Sequence

<220> <223> VH FR3 Re_Y

<400> 30 gccctggccc caataatcca tcagaaaata gccatcctgg cgcgcgcaat aatataccgc 60

<210> 31 <211> 54 <212> DNA <213> Artificial Sequence

<220> <223> VH Final Fo

<400> 31 ggttctggtg gtggtggttc tgctagcgac gtggtgatga cacagacgcc gctg 54

<210> 32 <211> 48 <212> DNA <213> Artificial Sequence

<220> <223> VH Final Re

<400> 32 ggagctcaca gtcaccagcg tgccctggcc ccaataatcc atcagaaa 48

<210> 33 <211> 27 <212> DNA <213> Artificial Sequence

<220> <223> VL FR1 Fo

<400> 33 gacgtggtga tgacacagac gccgctg 26 Feb 2019

27

<210> 34 <211> 57 <212> DNA <213> Artificial Sequence

<220> <223> VL FR1 Re_S 2019201141

<400> 34 gagccaattc agatacgtct tgccgtcgga gtccagcagc gactggcttg atttgca 57

<210> 35 <211> 57 <212> DNA <213> Artificial Sequence

<220> <223> VL FR1 Re_I

<400> 35 gagccaattc agatacgtct tgccgtcaat gtccagcagc gactggcttg atttgca 57

<210> 36 <211> 57 <212> DNA <213> Artificial Sequence

<220> <223> VL FR1 Re_L

<400> 36 gagccaattc agatacgtct tgccgtcaag gtccagcagc gactggcttg atttgca 57

<210> 37 <211> 57 <212> DNA <213> Artificial Sequence

<220> <223> VL FR1 Re_N

<400> 37 gagccaattc agatacgtct tgccgtcagc gtccagcagc gactggcttg atttgca 57

<210> 38 <211> 57 <212> DNA <213> Artificial Sequence

<220> 26 Feb 2019

<223> VL FR1 Re_Q

<400> 38 gagccaattc agatacgtct tgccgtcttg gtccagcagc gactggcttg atttgca 57

<210> 39 <211> 57 <212> DNA <213> Artificial Sequence 2019201141

<220> <223> VL FR1 Re_R

<400> 39 gagccaattc agatacgtct tgccgtctct gtccagcagc gactggcttg atttgca 57

<210> 40 <211> 57 <212> DNA <213> Artificial Sequence

<220> <223> VL FR1 Re_F

<400> 40 gagccaattc agatacgtct tgccgtcaaa gtccagcagc gactggcttg atttgca 57

<210> 41 <211> 57 <212> DNA <213> Artificial Sequence

<220> <223> VL FR1 Re_K

<400> 41 gagccaattc agatacgtct tgccgtcttt gtccagcagc gactggcttg atttgca 57

<210> 42 <211> 57 <212> DNA <213> Artificial Sequence

<220> <223> VL FR1 Re_T

<400> 42 gagccaattc agatacgtct tgccgtcagt gtccagcagc gactggcttg atttgca 57

<210> 43 <211> 57 26 Feb 2019

<212> DNA <213> Artificial Sequence

<220> <223> VL FR1 Re_V

<400> 43 gagccaattc agatacgtct tgccgtcaac gtccagcagc gactggcttg atttgca 57 2019201141

<210> 44 <211> 30 <212> DNA <213> Artificial Sequence

<220> <223> VL FR2 Fo

<400> 44 gacggcaaga cgtatctgaa ttggctccag 30

<210> 45 <211> 75 <212> DNA <213> Artificial Sequence

<220> <223> VL FR2 Re_T-YH

<400> 45 gcgtttaatt tcaaccttag tgccttggcc gaacgtaaac ggaaagtrgg tgccctgcca 60

gcaatagtag acgcc 75

<210> 46 <211> 75 <212> DNA <213> Artificial Sequence

<220> <223> VL FR2 Re_T-LIHQNK

<400> 46 gcgtttaatt tcaaccttag tgccttggcc gaacgtaaac ggaaawwkgg tgccctgcca 60

gcaatagtag acgcc 75

<210> 47 <211> 75 <212> DNA

<213> Artificial Sequence 26 Feb 2019

<220> <223> VL FR2 Re_FYIN-YH

<400> 47 gcgtttaatt tcaaccttag tgccttggcc gaacgtaaac ggaaagtraw wgccctgcca 60

gcaatagtag acgcc 75 2019201141

<210> 48 <211> 75 <212> DNA <213> Artificial Sequence

<220> <223> VL FR2 Re_FYIN-LIHQNK

<400> 48 gcgtttaatt tcaaccttag tgccttggcc gaacgtaaac ggaaawwkaw wgccctgcca 60

gcaatagtag acgcc 75

<210> 49 <211> 39 <212> DNA <213> Artificial Sequence

<220> <223> VL Final Re

<400> 49 gcgtttaatt tcaaccttag tgccttggcc gaacgtaaa 39

<210> 50 <211> 43 <212> DNA <213> Artificial Sequence

<220> <223> VL Final Fo SfiI

<400> 50 cgtggcccag gcggccgacg tggtgatgac acagacgccg ctg 43

<210> 51 <211> 33 <212> DNA <213> Artificial Sequence

<220>

<223> VL Final Fo NruI 26 Feb 2019

<400> 51 ctatcgcgat tgcagtggca ctggctggtt tcg 33

<210> 52 <211> 96 <212> DNA <213> Artificial Sequence

<220> 2019201141

<223> VL Overlapping Fo

<400> 52 ggcacgctgg tgactgtgag ctccggaggc ggcggaagtg gcggaggagg cagcggcgga 60

ggcgggagtg acgtggtgat gacacagacg ccgctg 96

<210> 53 <211> 72 <212> DNA <213> Artificial Sequence

<220> <223> VL Final Re

<400> 53 gtcctcttca gaaataagct tttgttcgga tccgcgttta atttcaacct tagtgccttg 60

gccgaacgta aa 72

<210> 54 <211> 66 <212> DNA <213> Artificial Sequence

<220> <223> VH Homologous recombination

<400> 54 gctctgcagg ctagtggtgg tggtggttct ggtggtggtg gttctggtgg tggtggttct 60

gctagc 66

<210> 55 <211> 60 <212> DNA <213> Artificial Sequence

<220>

<223> VL Homologous recombination 26 Feb 2019

<400> 55 ttgttatcag atctcgagct attacaagtc ctcttcagaa ataagctttt gttcggatcc 60

<210> 56 <211> 118 <212> PRT <213> Artificial Sequence

<220> 2019201141

<223> Clone 1001_Variable heavy chain

<400> 56

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Asp Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 57 <211> 113 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1001_Variable light chain

<400> 57

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ile 20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser 35 40 45

Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 2019201141

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95

Thr His Phe Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110

Arg

<210> 58 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1015_Variable heavy chain

<400> 58

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95 26 Feb 2019

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 59 <211> 118 2019201141

<212> PRT <213> Artificial Sequence

<220> <223> Clone 1021_Variable heavy chain

<400> 59

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Asp Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly His Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 60 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1023_Variable heavy chain

<400> 60 26 Feb 2019

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 2019201141

Gly Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Asp Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 61 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1024_Variable heavy chain

<400> 61

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Asp Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80 26 Feb 2019

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115 2019201141

<210> 62 <211> 113 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1024_Variable light chain

<400> 62

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Leu 20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser 35 40 45

Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95

Thr His Phe Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110

Arg

<210> 63 <211> 118 <212> PRT <213> Artificial Sequence

<220> 26 Feb 2019

<223> Clone 1104_Variable heavy chain

<400> 63

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 2019201141

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Gly Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 64 <211> 113 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1104_Variable light chain

<400> 64

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Val 20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser 35 40 45

Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60 26 Feb 2019

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95

Thr His Phe Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 2019201141

Arg

<210> 65 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1123_Variable heavy chain

<400> 65

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly His Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 66 <211> 118 26 Feb 2019

<212> PRT <213> Artificial Sequence

<220> <223> Clone 1202_Variable heavy chain

<400> 66

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 2019201141

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Gly Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Asp Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Lys Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 67 <211> 113 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1208_Variable light chain

<400> 67

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Val 20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser

35 40 45 26 Feb 2019

Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 2019201141

Thr Tyr Leu Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110

Arg

<210> 68 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1216_Variable heavy chain

<400> 68

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Tyr 20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly His Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115 26 Feb 2019

<210> 69 <211> 113 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1216_Variable light chain

<400> 69 2019201141

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ile 20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser 35 40 45

Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95

Thr His Leu Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110

Arg

<210> 70 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1223_Variable heavy chain

<400> 70

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30 26 Feb 2019

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 2019201141

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly His Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 71 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1234_Variable heavy chain

<400> 71

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 26 Feb 2019

Leu Val Thr Val Ser Ser 115

<210> 72 <211> 113 <212> PRT <213> Artificial Sequence 2019201141

<220> <223> Clone 1234_Variable light chain

<400> 72

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ile 20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser 35 40 45

Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Glu Ile 65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95

Thr His Phe Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110

Arg

<210> 73 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1243_Variable heavy chain

<400> 73

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15 26 Feb 2019

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val 50 55 60 2019201141

His Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly His Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 74 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1248_Variable heavy chain

<400> 74

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Asp Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 26 Feb 2019

Ala Arg Gln Asp Gly His Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 75 2019201141

<211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 3007_Variable heavy chain

<400> 75

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr 20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Leu Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 76 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 3016_Variable heavy chain

<400> 76 26 Feb 2019

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ser Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 77 <211> 113 <212> PRT <213> Artificial Sequence

<220> <223> Clone 3016_Variable light chain

<400> 77

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser 35 40 45

Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 26 Feb 2019

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95

Thr His Leu Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110

Arg 2019201141

<210> 78 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 3024_Variable heavy chain

<400> 78

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 79 <211> 118 <212> PRT

<213> Artificial Sequence 26 Feb 2019

<220> <223> Clone 3120_Variable heavy chain

<400> 79

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Tyr Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 80 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 3131_Variable heavy chain

<400> 80

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 26 Feb 2019

Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Gln Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 2019201141

100 105 110

Leu Val Thr Val Ser Ser 115

<210> 81 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 3203_Variable heavy chain

<400> 81

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 82 26 Feb 2019

<211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 4011_Variable heavy chain

<400> 82

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Tyr Ser Tyr 20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 83 <211> 113 <212> PRT <213> Artificial Sequence

<220> <223> Clone 4011_Variable light chain

<400> 83

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Val 20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser 35 40 45 26 Feb 2019

Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 2019201141

85 90 95

Thr His Leu Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110

Arg

<210> 84 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 4017_Variable heavy chain

<400> 84

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Gly Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Tyr Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 26 Feb 2019

115

<210> 85 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 4034_Variable heavy chain 2019201141

<400> 85

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Tyr Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 86 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 4041_Variable heavy chain

<400> 86

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 26 Feb 2019

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 2019201141

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Tyr Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 87 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 4141_Variable heavy chain

<400> 87

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Gly Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Tyr Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 26 Feb 2019

100 105 110

Leu Val Thr Val Ser Ser 115

<210> 88 <211> 118 <212> PRT <213> Artificial Sequence 2019201141

<220> <223> Clone 4146_Variable heavy chain

<400> 88

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Tyr Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 89 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 4206_Variable heavy chain

<400> 89

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 26 Feb 2019

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val 2019201141

50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asp Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 90 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 4208_Variable heavy chain

<400> 90

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Ser Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Gly Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 26 Feb 2019

85 90 95

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115 2019201141

<210> 91 <211> 113 <212> PRT <213> Artificial Sequence

<220> <223> Clone 4208_Variable light chain

<400> 91

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Thr 20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser 35 40 45

Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95

Thr His Phe Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110

Arg

<210> 92 <211> 120 <212> PRT <213> Artificial Sequence

<220>

<223> Clone 4278_Variable heavy chain 26 Feb 2019

<400> 92

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr 20 25 30

Ala Met Asn Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 2019201141

35 40 45

Ser Thr Ile Thr Leu Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Asp Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Tyr Leu Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln 100 105 110

Gly Thr Leu Val Thr Val Ser Ser 115 120

<210> 93 <211> 120 <212> PRT <213> Artificial Sequence

<220> <223> Clone 4287_Variable heavy chain

<400> 93

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr 20 25 30

Ala Met Asn Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 26 Feb 2019

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln His Pro Tyr Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln 100 105 110 2019201141

Gly Thr Leu Val Thr Val Ser Ser 115 120

<210> 94 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 4_Variable heavy chain

<400> 94

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Gly Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 95 <211> 118

<212> PRT <213> Artificial Sequence 26 Feb 2019

<220> <223> Clone 6_Variable heavy chain

<400> 95

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 2019201141

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Gly Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 96 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 9_Variable heavy chain

<400> 96

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Gly Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 26 Feb 2019

50 55 60

Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 2019201141

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 97 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 11_Variable heavy chain

<400> 97

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Gly Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 98 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 12_Variable heavy chain

<400> 98

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 2019201141

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Gly Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly His Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 99 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 13_Variable heavy chain

<400> 99

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 26 Feb 2019

35 40 45

Gly Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 100 <211> 113 <212> PRT <213> Artificial Sequence

<220> <223> Clone 14_Variable light chain

<400> 100

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Pro Ser Leu Leu Asp Ser 20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser 35 40 45

Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95

Thr Tyr Phe Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110

Arg

<210> 101 <211> 113 <212> PRT <213> Artificial Sequence

<220> <223> Clone 15_Variable light chain 2019201141

<400> 101

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Pro Ser Leu Leu Asp Ser 20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser 35 40 45

Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95

Phe Tyr Phe Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110

Arg

<210> 102 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 16_Variable heavy chain

<400> 102

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Tyr 26 Feb 2019

20 25 30

Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 103 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 17_Variable heavy chain

<400> 103

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 104 <211> 118 <212> PRT 2019201141

<213> Artificial Sequence

<220> <223> Clone 18_Variable heavy chain

<400> 104

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Tyr 20 25 30

Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Asp Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 105 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 19_Variable heavy chain

<400> 105

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 26 Feb 2019

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gln Tyr 20 25 30

Ser Thr Ile Thr Lys Lys Gly Ser Phe Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Asp Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Glu Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 106 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 20_Variable heavy chain

<400> 106

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gln Tyr 20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Lys Lys Gly Gly Ser Phe Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Asp Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Glu Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115 2019201141

<210> 107 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 21_Variable heavy chain

<400> 107

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Lys Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Asp Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 108 <211> 113 <212> PRT <213> Artificial Sequence

<220> <223> Clone 22_Variable light chain 26 Feb 2019

<400> 108

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Pro Ser Leu Leu Asp Val 20 25 30 2019201141

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser 35 40 45

Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95

Thr Tyr Phe Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110

Arg

<210> 109 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 23_Variable heavy chain

<400> 109

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Tyr 20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly His Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 2019201141

Leu Val Thr Val Ser Ser 115

<210> 110 <211> 5 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1001_Variable heavy chain CDR 1

<400> 110

Ser Tyr Ala Met Asn 1 5

<210> 111 <211> 17 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1001_Variable heavy chain CDR 2

<400> 111

Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val Asp 1 5 10 15

Gly

<210> 112 <211> 9 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1001_Variable heavy chain CDR 3

<400> 112

Gln Asp Gly Asn Phe Leu Met Asp Tyr

1 5 26 Feb 2019

<210> 113 <211> 16 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1001_Variable light chain CDR 1

<400> 113 2019201141

Lys Ser Ser Gln Ser Leu Leu Asp Ile Asp Gly Lys Thr Tyr Leu Asn 1 5 10 15

<210> 114 <211> 7 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1001_Variable light chain CDR 2

<400> 114

Leu Val Ser Lys Leu Asp Ser 1 5

<210> 115 <211> 8 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1001_Variable light chain CDR 3

<400> 115

Trp Gln Gly Thr His Phe Pro Phe 1 5

<210> 116 <211> 17 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1015_Variable heavy chain CDR 2

<400> 116

Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val Glu 1 5 10 15

Gly

<210> 117 <211> 9 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1021_Variable heavy chain CDR 3

<400> 117

Gln Asp Gly His Phe Leu Met Asp Tyr 2019201141

1 5

<210> 118 <211> 5 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1024_Variable heavy chain CDR 1

<400> 118

Ser Tyr Ala Met Ser 1 5

<210> 119 <211> 16 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1024_Variable light chain CDR 1

<400> 119

Lys Ser Ser Gln Ser Leu Leu Asp Leu Asp Gly Lys Thr Tyr Leu Asn 1 5 10 15

<210> 120 <211> 17 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1104_Variable heavy chain CDR 2

<400> 120

Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val Gln 1 5 10 15

Gly

<210> 121 26 Feb 2019

<211> 16 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1104_Variable light chain CDR 1

<400> 121

Lys Ser Ser Gln Ser Leu Leu Asp Val Asp Gly Lys Thr Tyr Leu Asn 1 5 10 15 2019201141

<210> 122 <211> 8 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1208_Variable light chain CDR 3

<400> 122

Trp Gln Gly Thr Tyr Leu Pro Phe 1 5

<210> 123 <211> 17 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1214_Variable heavy chain CDR 2

<400> 123

Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val Glu 1 5 10 15

Gly

<210> 124 <211> 5 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1216_Variable heavy chain CDR 1

<400> 124

His Tyr Ala Met Asn 1 5

<210> 125 <211> 8 26 Feb 2019

<212> PRT <213> Artificial Sequence

<220> <223> Clone 1216_Variable light chain CDR 3

<400> 125

Trp Gln Gly Thr His Leu Pro Phe 1 5 2019201141

<210> 126 <211> 17 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1234_Variable heavy chain CDR 2

<400> 126

Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val Gln 1 5 10 15

Gly

<210> 127 <211> 17 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1243_Variable heavy chain CDR 2

<400> 127

Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val His 1 5 10 15

Gly

<210> 128 <211> 17 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1248_Variable heavy chain CDR 2

<400> 128

Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val Asp

1 5 10 15 26 Feb 2019

Gly

<210> 129 <211> 17 <212> PRT <213> Artificial Sequence

<220> 2019201141

<223> Clone 3007_Variable heavy chain CDR 2

<400> 129

Thr Ile Thr Leu Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val Gln 1 5 10 15

Gly

<210> 130 <211> 16 <212> PRT <213> Artificial Sequence

<220> <223> Clone 3016_Variable light chain CDR 1

<400> 130

Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn 1 5 10 15

<210> 131 <211> 9 <212> PRT <213> Artificial Sequence

<220> <223> Clone 3120_Variable heavy chain CDR 3

<400> 131

Gln Asp Gly Tyr Phe Leu Met Asp Tyr 1 5

<210> 132 <211> 9 <212> PRT <213> Artificial Sequence

<220> <223> Clone 3131_Variable heavy chain CDR 3

<400> 132 26 Feb 2019

Gln Asp Gly Gln Phe Leu Met Asp Tyr 1 5

<210> 133 <211> 5 <212> PRT <213> Artificial Sequence

<220> <223> Clone 4278_Variable heavy chain CDR 1 2019201141

<400> 133

Lys Tyr Ala Met Asn 1 5

<210> 134 <211> 17 <212> PRT <213> Artificial Sequence

<220> <223> Clone 4278_Variable heavy chain CDR 2

<400> 134

Thr Ile Thr Leu Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val Asp 1 5 10 15

Gly

<210> 135 <211> 11 <212> PRT <213> Artificial Sequence

<220> <223> Clone 4278_Variable heavy chain CDR 3

<400> 135

Gln Tyr Leu Asp Gly Asn Phe Leu Met Asp Tyr 1 5 10

<210> 136 <211> 11 <212> PRT <213> Artificial Sequence

<220> <223> Clone 4287_Variable heavy chain CDR 3

<400> 136

Gln His Pro Tyr Gly Asn Phe Leu Met Asp Tyr 26 Feb 2019

1 5 10

<210> 137 <211> 16 <212> PRT <213> Artificial Sequence

<220> <223> Clone 1_Variable light chain CDR 1 2019201141

<400> 137

Lys Ser Ser Pro Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn 1 5 10 15

<210> 138 <211> 16 <212> PRT <213> Artificial Sequence

<220> <223> Clone 2_Variable light chain CDR 1

<400> 138

Lys Ser Ser Pro Ser Leu Leu Asp Ile Asp Gly Lys Thr Tyr Leu Asn 1 5 10 15

<210> 139 <211> 16 <212> PRT <213> Artificial Sequence

<220> <223> Clone 3_Variable light chain CDR 1

<400> 139

Lys Ser Ser Pro Ser Leu Leu Asp Val Asp Gly Lys Thr Tyr Leu Asn 1 5 10 15

<210> 140 <211> 8 <212> PRT <213> Artificial Sequence

<220> <223> Clone 14_Variable light chain CDR 3

<400> 140

Trp Gln Gly Thr Tyr Phe Pro Phe 1 5

<210> 141 <211> 8 26 Feb 2019

<212> PRT <213> Artificial Sequence

<220> <223> Clone 15_Variable light chain CDR 3

<400> 141

Trp Gln Gly Phe Tyr Phe Pro Phe 1 5 2019201141

<210> 142 <211> 5 <212> PRT <213> Artificial Sequence

<220> <223> Clone 16_Variable heavy chain CDR 1

<400> 142

His Tyr Ala Met Thr 1 5

<210> 143 <211> 5 <212> PRT <213> Artificial Sequence

<220> <223> Clone 17_Variable heavy chain CDR 1

<400> 143

Ser Tyr Ala Met Thr 1 5

<210> 144 <211> 5 <212> PRT <213> Artificial Sequence

<220> <223> Clone 19_Variable heavy chain CDR 1

<400> 144

Gln Tyr Ala Met Asn 1 5

<210> 145 <211> 17 <212> PRT <213> Artificial Sequence

<220> <223> Clone 19_Variable heavy chain CDR 2 26 Feb 2019

<400> 145

Thr Ile Thr Lys Lys Gly Ser Phe Thr Tyr Tyr Ala Asp Ser Val Asp 1 5 10 15

Gly 2019201141

<210> 146 <211> 9 <212> PRT <213> Artificial Sequence

<220> <223> Clone 19_Variable heavy chain CDR 3

<400> 146

Gln Asp Gly Glu Phe Leu Met Asp Tyr 1 5

<210> 147 <211> 17 <212> PRT <213> Artificial Sequence

<220> <223> Clone 20_Variable heavy chain CDR 2

<400> 147

Thr Ile Lys Lys Gly Gly Ser Phe Thr Tyr Tyr Ala Asp Ser Val Asp 1 5 10 15

Gly

<210> 148 <211> 17 <212> PRT <213> Artificial Sequence

<220> <223> Clone 21_Variable heavy chain CDR 2

<400> 148

Thr Ile Thr Lys Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val Asp 1 5 10 15

Gly

<210> 149 <211> 5 <212> PRT <213> Artificial Sequence

<220> <223> Clone 23_Variable heavy chain CDR 1

<400> 149

His Tyr Ala Met Asn 2019201141

1 5

<210> 150 <211> 53 <212> DNA <213> Artificial Sequence

<220> <223> VH Fo

<400> 150 tgctgtgggt gagtggtacc tgtggggaag tccagctggt ggagtctgga ggt 53

<210> 151 <211> 56 <212> DNA <213> Artificial Sequence

<220> <223> VH Re

<400> 151 agtgggaaca cggagggccc cttggtgctg gcggagctca cagtcaccag cgtgcc 56

<210> 152 <211> 53 <212> DNA <213> Artificial Sequence

<220> <223> VL Fo

<400> 152 tgctgtgggt gagtggtacc tgtggggacg tggtgatgac acagacgccg ctg 53

<210> 153 <211> 66 <212> DNA <213> Artificial Sequence

<220>

<223> VL Re_CL overlap 26 Feb 2019

<400> 153 gatgaacaca gaaggggcag ccaccgtgcg tttaatttca accttagtgc cttggccgaa 60

cgtaaa 66

<210> 154 <211> 27 <212> DNA 2019201141

<213> Artificial Sequence

<220> <223> Ck Fo

<400> 154 acggtggctg ccccttctgt gttcatc 27

<210> 155 <211> 45 <212> DNA <213> Artificial Sequence

<220> <223> Ck Re

<400> 155 gattggatcc aagcttacta gcactcaccc ctgttgaaag actta 45

<210> 156 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone A24_Variable heavy chain

<400> 156

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe His Ser Tyr 20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Thr Arg Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 26 Feb 2019

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 2019201141

Leu Val Thr Val Ser Ser 115

<210> 157 <211> 113 <212> PRT <213> Artificial Sequence

<220> <223> Clone A25_Variable light chain

<400> 157

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Arg 20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser 35 40 45

Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95

Thr His Phe Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110

Arg

<210> 158 <211> 118

<212> PRT <213> Artificial Sequence 26 Feb 2019

<220> <223> Clone A52_Variable heavy chain

<400> 158

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 2019201141

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Thr Gly Gly Ser His Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Asp Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 159 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone A63_Variable heavy chain

<400> 159

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 26 Feb 2019

50 55 60

Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 160 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone A71_Variable heavy chain

<400> 160

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60

His Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 161 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone A74_Variable heavy chain

<400> 161

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 2019201141

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 162 <211> 16 <212> PRT <213> Artificial Sequence

<220> <223> Clone A25_Variable light chain CDR 1

<400> 162

Lys Ser Ser Gln Ser Leu Leu Asp Arg Asp Gly Lys Thr Tyr Leu Asn 1 5 10 15

<210> 163 <211> 17 <212> PRT

<213> Artificial Sequence 26 Feb 2019

<220> <223> Clone A71_Variable heavy chain CDR 2

<400> 163

Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val His 1 5 10 15

Gly 2019201141

<210> 164 <211> 5 <212> PRT <213> Artificial Sequence

<220> <223> Clone A74_Variable heavy chain CDR 1

<400> 164

Ser Tyr Ala Met His 1 5

<210> 165 <211> 304 <212> PRT <213> Artificial Sequence

<220> <223> HUMAN Tissue factor pathway inhibitor

<400> 165

Met Ile Tyr Thr Met Lys Lys Val His Ala Leu Trp Ala Ser Val Cys 1 5 10 15

Leu Leu Leu Asn Leu Ala Pro Ala Pro Leu Asn Ala Asp Ser Glu Glu 20 25 30

Asp Glu Glu His Thr Ile Ile Thr Asp Thr Glu Leu Pro Pro Leu Lys 35 40 45

Leu Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys 50 55 60

Ala Ile Met Lys Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu 65 70 75 80

Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser 85 90 95

Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp Asn Ala Asn Arg Ile 100 105 110

Ile Lys Thr Thr Leu Gln Gln Glu Lys Pro Asp Phe Cys Phe Leu Glu 115 120 125

Glu Asp Pro Gly Ile Cys Arg Gly Tyr Ile Thr Arg Tyr Phe Tyr Asn 130 135 140 2019201141

Asn Gln Thr Lys Gln Cys Glu Arg Phe Lys Tyr Gly Gly Cys Leu Gly 145 150 155 160

Asn Met Asn Asn Phe Glu Thr Leu Glu Glu Cys Lys Asn Ile Cys Glu 165 170 175

Asp Gly Pro Asn Gly Phe Gln Val Asp Asn Tyr Gly Thr Gln Leu Asn 180 185 190

Ala Val Asn Asn Ser Leu Thr Pro Gln Ser Thr Lys Val Pro Ser Leu 195 200 205

Phe Glu Phe His Gly Pro Ser Trp Cys Leu Thr Pro Ala Asp Arg Gly 210 215 220

Leu Cys Arg Ala Asn Glu Asn Arg Phe Tyr Tyr Asn Ser Val Ile Gly 225 230 235 240

Lys Cys Arg Pro Phe Lys Tyr Ser Gly Cys Gly Gly Asn Glu Asn Asn 245 250 255

Phe Thr Ser Lys Gln Glu Cys Leu Arg Ala Cys Lys Lys Gly Phe Ile 260 265 270

Gln Arg Ile Ser Lys Gly Gly Leu Ile Lys Thr Lys Arg Lys Arg Lys 275 280 285

Lys Gln Arg Val Lys Ile Ala Tyr Glu Glu Ile Phe Val Lys Asn Met 290 295 300

<210> 166 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone T417_mouse Variable heavy chain

<400> 166 26 Feb 2019

Glu Val His Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val 35 40 45 2019201141

Ala Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80

Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Thr Val Thr Val Ser Ser 115

<210> 167 <211> 113 <212> PRT <213> Artificial Sequence

<220> <223> Clone T417_mouse Variable light chain

<400> 167

Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45

Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95

Thr His Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110

Arg 2019201141

<210> 168 <211> 120 <212> PRT <213> Artificial Sequence

<220> <223> Clone T308_mouse Variable heavy chain

<400> 168

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30

Pro Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45

Ala Thr Ile Ser Asn Ser Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95

Ala Arg Gln Val Tyr Gly Asn Tyr Glu Asp Phe Asp Tyr Trp Gly Gln 100 105 110

Gly Thr Thr Leu Thr Val Ser Ser 115 120

<210> 169 <211> 113 <212> PRT

<213> Artificial Sequence 26 Feb 2019

<220> <223> Clone T308_mouse Variable light chain

<400> 169

Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 2019201141

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45

Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95

Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys 100 105 110

Arg

<210> 170 <211> 5 <212> PRT <213> Artificial Sequence

<220> <223> Clone T417_mouse Variable heavy chain CDR 1

<400> 170

Ser Tyr Ala Met Ser 1 5

<210> 171 <211> 17 <212> PRT <213> Artificial Sequence

<220> <223> Clone T417_mouse Variable heavy chain CDR 2

<400> 171 26 Feb 2019

Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Lys 1 5 10 15

Gly

<210> 172 <211> 9 <212> PRT 2019201141

<213> Artificial Sequence

<220> <223> Gln Asp Gly Asn Phe Leu Met Asp Tyr

<400> 172

Gln Asp Gly Asn Phe Leu Met Asp Tyr 1 5

<210> 173 <211> 16 <212> PRT <213> Artificial Sequence

<220> <223> Clone T417_mouse Variable light chain CDR 1

<400> 173

Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn 1 5 10 15

<210> 174 <211> 7 <212> PRT <213> Artificial Sequence

<220> <223> Clone T417_mouse Variable light chain CDR 2

<400> 174

Leu Val Ser Lys Leu Asp Ser 1 5

<210> 175 <211> 8 <212> PRT <213> Artificial Sequence

<220> <223> Clone T417_mouse Variable light chain CDR 3

<400> 175

Trp Gln Gly Thr His Phe Pro Phe 26 Feb 2019

1 5

<210> 176 <211> 5 <212> PRT <213> Artificial Sequence

<220> <223> Clone T308_mouse Variable heavy chain CDR 1 2019201141

<400> 176

Asn Tyr Pro Met Ser 1 5

<210> 177 <211> 17 <212> PRT <213> Artificial Sequence

<220> <223> Clone T308_mouse Variable heavy chain CDR 2

<400> 177

Thr Ile Ser Asn Ser Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Lys 1 5 10 15

Gly

<210> 178 <211> 11 <212> PRT <213> Artificial Sequence

<220> <223> Clone T308_mouse Variable heavy chain CDR 3

<400> 178

Gln Val Tyr Gly Asn Tyr Glu Asp Phe Asp Tyr 1 5 10

<210> 179 <211> 16 <212> PRT <213> Artificial Sequence

<220> <223> Clone T308_mouse Variable light chain CDR 1

<400> 179

Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn 1 5 10 15 26 Feb 2019

<210> 180 <211> 7 <212> PRT <213> Artificial Sequence

<220> <223> Clone T308_mouse Variable light chain CDR 2

<400> 180 2019201141

Leu Val Ser Lys Leu Asp Ser 1 5

<210> 181 <211> 8 <212> PRT <213> Artificial Sequence

<220> <223> Clone T308_mouse Variable light chain CDR 3

<400> 181

Trp Gln Gly Thr His Phe Pro Tyr 1 5

<210> 182 <211> 87 <212> DNA <213> Artificial Sequence

<220> <223> Primer_T417VH-F

<400> 182 gcggccgcca tgtatctggg tctgaactat gtctttatcg tgtttctgct gaatggtgtg 60

cagtctgagg tgcacctggt ggagtct 87

<210> 183 <211> 43 <212> DNA <213> Artificial Sequence

<220> <223> Primer_T417VH Apa-R

<400> 183 nnnngggccc cttggtgctg gctgaggaga cggtgaccgt ggt 43

<210> 184 26 Feb 2019

<211> 95 <212> DNA <213> Artificial Sequence

<220> <223> Primer_T417 VL-F

<400> 184 gcggccgcca tggatagcca ggctcaggtg ctgatgctgc tgctgctgtg ggtgtcaggg 60 2019201141

acttgcgggg acgttgtgat gacccagact ccact 95

<210> 185 <211> 31 <212> DNA <213> Artificial Sequence

<220> <223> Primer_VL-R

<400> 185 nnnnggtacc agatttcaac tgctcatcag a 31

<210> 186 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 308_humanized Variable heavy chain

<400> 186

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 187 2019201141

<211> 113 <212> PRT <213> Artificial Sequence

<220> <223> Clone 308_humanized Variable light chain

<400> 187

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser 35 40 45

Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95

Thr His Phe Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110

Arg

<210> 188 <211> 5 <212> PRT <213> Artificial Sequence

<220> <223> Clone 308_humanized Variable heavy chain CDR 1

<400> 188 26 Feb 2019

Ser Tyr Ala Met Asn 1 5

<210> 189 <211> 118 <212> PRT <213> Artificial Sequence

<220> 2019201141

<223> Clone 308-2_humanized and mutated Variable heavy chain

<400> 189

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60

Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 190 <211> 118 <212> PRT <213> Artificial Sequence

<220> <223> Clone 308-4_humanized and mutated Variable heavy chain

<400> 190

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 2019201141

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gln Asp Gly Asn Phe Leu Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110

Leu Val Thr Val Ser Ser 115

<210> 191 <211> 17 <212> PRT <213> Artificial Sequence

<220> <223> Clone 308-2_humanized and mutated Variable heavy chain CDR 2

<400> 191

Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Gln 1 5 10 15

Gly

<210> 192 <211> 17 <212> PRT <213> Artificial Sequence

<220> <223> Clone 308-4_humanized and mutated Variable heavy chain CDR 2

<400> 192

Thr Ile Thr Thr Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Glu 1 5 10 15

Gly 26 Feb 2019

<210> 193 <211> 50 <212> DNA <213> Artificial Sequence

<220> <223> Primer_VH Fo 2019201141

<400> 193 tgctgtgggt gagtggtacc tgtggggaag tgcagctcgt ggagagcggt 50

<210> 194 <211> 56 <212> DNA <213> Artificial Sequence

<220> <223> Primer_VH Re

<400> 194 agtgggaaca cggagggccc cttggtgctg gcggatgaga cagtcacaag tgtccc 56

<210> 195 <211> 58 <212> PRT <213> Artificial Sequence

<220> <223> Human_TFPI Kunitz domain 2

<400> 195

Lys Pro Asp Phe Cys Phe Leu Glu Glu Asp Pro Gly Ile Cys Arg Gly 1 5 10 15

Tyr Ile Thr Arg Tyr Phe Tyr Asn Asn Gln Thr Lys Gln Cys Glu Arg 20 25 30

Phe Lys Tyr Gly Gly Cys Leu Gly Asn Met Asn Asn Phe Glu Thr Leu 35 40 45

Glu Glu Cys Lys Asn Ile Cys Glu Asp Gly 50 55

<210> 196 <211> 58 <212> PRT <213> Artificial Sequence

<220> 26 Feb 2019

<223> Rabbit_TFPI Kunitz domain 2

<400> 196

Lys Pro Asp Phe Cys Phe Leu Glu Glu Asp Pro Gly Ile Cys Arg Gly 1 5 10 15

Phe Met Thr Arg Tyr Phe Tyr Asn Asn Gln Ser Lys Gln Cys Glu Gln 20 25 30 2019201141

Phe Lys Tyr Gly Gly Cys Leu Gly Asn Ser Asn Asn Phe Glu Thr Leu 35 40 45

Glu Glu Cys Arg Asn Thr Cys Glu Asp Pro 50 55

<210> 197 <211> 58 <212> PRT <213> Artificial Sequence

<220> <223> Mouse_TFPI Kunitz domain 2

<400> 197

Arg Pro Asp Phe Cys Phe Leu Glu Glu Asp Pro Gly Leu Cys Arg Gly 1 5 10 15

Tyr Met Lys Arg Tyr Leu Tyr Asn Asn Gln Thr Lys Gln Cys Glu Arg 20 25 30

Phe Val Tyr Gly Gly Cys Leu Gly Asn Arg Asn Asn Phe Glu Thr Leu 35 40 45

Asp Glu Cys Lys Lys Ile Cys Glu Asn Pro 50 55

<210> 198 <211> 29 <212> DNA <213> Artificial Sequence

<220> <223> Primer_HTK2 For

<400> 198 ccatggaaac ccgacttttg cttcctgga 29

<210> 199 <211> 30 26 Feb 2019

<212> DNA <213> Artificial Sequence

<220> <223> Primer_RTK2 For

<400> 199 ccatggaaac ccgatttctg ctttctggag 30 2019201141

<210> 200 <211> 30 <212> DNA <213> Artificial Sequence

<220> <223> Primer_MTK2 For

<400> 200 ccatggagac ctgacttctg ctttctggag 30

<210> 201 <211> 33 <212> DNA <213> Artificial Sequence

<220> <223> Primer_HTK2 Re

<400> 201 gcggccgcct agccgtcttc acagatgttc ttg 33

<210> 202 <211> 30 <212> DNA <213> Artificial Sequence

<220> <223> Primer_RTK2 Re

<400> 202 gcggccgcct aggggtcctc acaggtgttg 30

<210> 203 <211> 37 <212> DNA <213> Artificial Sequence

<220> <223> Primer_MTK2 Re

<400> 203 gcggccgcct aggggttctc acagattttc ttgcatt 37 26 Feb 2019 2019201141

Claims

1. An antibody that binds specifically to a TFPI (tissue factor pathway inhibitor) represented by SEQ ID NO: 39, comprising a heavy-chain CDR1 comprising an amino acid sequence of SEQ ID NO: 5, a heavychain CDR2 comprising an amino acid sequence of SEQ ID NO: 6, and a heavy-chain CDR3 comprising an amino acid sequence of SEQ ID NO: 7; and a light-chain CDR1 comprising an amino acid sequence of SEQ ID NO: 8, a light-chain CDR2 comprising an amino acid sequence of SEQ ID NO: 9, and a light-chain CDR3 comprising an amino acid sequence of SEQ ID NO: 10;

a heavy-chain CDR1 comprising an amino acid sequence of SEQ ID NO: 23, a heavy-chain CDR2 comprising an amino acid sequence of SEQ ID NO: 6, and a heavychain CDR3 comprising an amino acid sequence of SEQ ID NO: 7; and a light-chain CDR1 comprising an amino acid sequence of SEQ ID NO: 8, a light-chain CDR2 comprising an amino acid sequence of SEQ ID NO: 9, and a light-chain CDR3 comprising an amino acid sequence of SEQ ID NO: 10;

a heavy-chain CDR1 comprising an amino acid sequence of SEQ ID NO: 23, a heavy-chain CDR2 comprising an amino acid sequence of SEQ ID NO: 27, and a heavychain CDR3 comprising an amino acid sequence of SEQ ID NO: 7; and a light-chain CDR1 comprising an amino acid sequence of SEQ ID NO: 8, a light-chain CDR2 comprising an amino acid sequence of SEQ ID NO: 9, and a light-chain CDR3 comprising an amino acid sequence of SEQ ID NO: 10;

a heavy-chain CDR1 comprising an amino acid sequence of SEQ ID NO: 149, a heavy-chain CDR2 comprising an amino acid sequence of SEQ ID NO: 162, and a heavychain CDR3 comprising an amino acid sequence of SEQ ID NO: 151; and a light-chain CDR1 comprising an amino acid sequence of SEQ ID NO: 177, a light-chain CDR2

136 comprising an amino acid sequence of SEQ ID NO: 153, and a light-chain CDR3 comprising an amino acid sequence of SEQ ID NO: 154;

a heavy-chain CDR1 comprising an amino acid sequence of SEQ ID NO: 149, a heavy-chain CDR2 comprising an amino acid sequence of SEQ ID NO: 155, and a heavychain CDR3 comprising an amino acid sequence of SEQ ID NO: 156; and a light-chain CDR1 comprising an amino acid sequence of SEQ ID NO: 177, a light-chain CDR2 comprising an amino acid sequence of SEQ ID NO: 153, and a light-chain CDR3 comprising an amino acid sequence of SEQ ID NO: 154;

a heavy-chain CDR1 comprising an amino acid sequence of SEQ ID NO: 149, a heavy-chain CDR2 comprising an amino acid sequence of SEQ ID NO: 155, and a heavychain CDR3 comprising an amino acid sequence of SEQ ID NO: 151; and a light-chain CDR1 comprising an amino acid sequence of SEQ ID NO: 177, a light-chain CDR2 comprising an amino acid sequence of SEQ ID NO: 153, and a light-chain CDR3 comprising an amino acid sequence of SEQ ID NO: 154; or a heavy-chain CDR1 comprising an amino acid sequence of SEQ ID NO: 149, a heavy-chain CDR2 comprising an amino acid sequence of SEQ ID NO: 162, and a heavychain CDR3 comprising an amino acid sequence of SEQ ID NO: 151; and a light-chain CDR1 comprising an amino acid sequence of SEQ ID NO: 152, a light-chain CDR2 comprising an amino acid sequence of SEQ ID NO: 153, and a light-chain CDR3 comprising an amino acid sequence of SEQ ID NO: 154.

2. The antibody of claim 1, wherein the antibody contains a heavy-chain variable region comprising a sequence having a homology of at least 80% to an amino acid sequence of SEQ ID NO: 1, 21, 25, 95, 105, 137, or 138.

137

2019201141 09 Apr 2020

3. The antibody of claim 1, the antibody contains a light-chain variable region comprising a sequence having a homology of at least 80% to an amino acid sequence of SEQ ID NO: 2 or 22.

4. A nucleic acid encoding the antibody of any one of claims 1 to 3.

5 5. A vector comprising the nucleic acid of claim 4.

6. A host cell comprising the vector of claim 5.

7. A method for producing the antibody of any one of claims 1 to 3, which comprises culturing the host cell of claim 6 to express the antibody.

8. A pharmaceutical composition for treating hemophilia, which comprises the

10 antibody of any one of claims 1 to 3 as an active ingredient.